Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis

ABSTRACT

Nucleotide triphosphate probes containing a molecular and/or atomic tag on a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/137,199, filed Dec. 20, 2013, now issued as U.S. Pat. No. 9,243,284;which is a continuation of U.S. patent application Ser. No. 13/644,469,filed Oct. 4, 2012, now issued as U.S. Pat. No. 8,648,179; which is acontinuation of U.S. patent application Ser. No. 11/648,721, filed Dec.29, 2006, now issued as U.S. Pat. No. 8,314,216; which is a continuationof U.S. patent application Ser. No. 10/007,621, filed Dec. 3, 2001, nowissued as U.S. Pat. No. 7,211,414; which claims priority to U.S.Provisional Patent Application Ser. No. 60/250,764, filed Dec. 1, 2000,all of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for alteringthe fidelity of nucleic acid synthesis.

More particularly, the present invention relates to the followinggeneral areas: (1) nucleotide triphosphate monomers having at least onemolecular or atomic tag bonded to and/or chemically and/or physicallyassociated with one or more of the phosphate groups of the triphosphatemoiety of the monomers, the base moiety, and/or the sugar moiety in thecase of a nucleoside analog; (2) methods for enzymatic DNA synthesiswith altered fidelity; (3) methods of sequencing DNA, based on thedetection of base incorporation using tags bonded to and/or chemicallyand/or physically associated with the β and/or γ phosphates of thetriphosphate of the nucleotide monomer, the base moiety of a nucleotideor nucleoside monomer, and/or the sugar moiety of a nucleotide ornucleoside monomer, the polymerase or by the release of the taggedpyrophosphate (PPi); (4) a template-mediated primer extension reactionwith improved monomer incorporation fidelity using the tagged monomers;(5) methods for performing a primer extension reaction, such as a DNAsequencing reaction, or a polymerase chain reaction using the taggedmonomers; (6) methods for improving nucleotide incorporation fidelity byadding tagged pyrophosphate (PP_(i)) to a monomer polymerization medium,where the monomers can be tagged or untagged; and (7) kits forconducting nucleotide sequencing, a polymerase chain reaction, atemplated-mediated primer extension reaction or similar reaction withimproved monomer incorporation fidelity using either taggedpyrophosphate and/or untagged or tagged monomers.

2. Description of the Related Art

Sequencing Nucleic Acids Using Tagged Monomers

The primary sequences of nucleic acids are crucial for understanding thefunction and control of genes and for applying many of the basictechniques of molecular biology. The ability to do rapid and reliableDNA sequencing is, therefore, a very important technology. The DNAsequence is an important tool in genomic analysis as well as otherapplications, such as genetic identification, forensic analysis, geneticcounseling, medical diagnostics, etc. With respect to the area ofmedical diagnostic sequencing, disorders, susceptibilities to disorders,and prognoses of disease conditions, can be correlated with the presenceof particular DNA sequences, or the degree of variation (or mutation) inDNA sequences, at one or more genetic loci. Examples of such phenomenainclude human leukocyte antigen (HLA) typing, cystic fibrosis, tumorprogression and heterogeneity, p53 proto-oncogene mutations and rasproto-oncogene mutations. See, e.g., Gyllensten et al., PCR Methods andApplications, 1: 91-98 (1991); U.S. Pat. No. 5,578,443, issued toSantamaria et al., incorporated herein by reference; and U.S. Pat. No.5,776,677, issued to Tsui et al., incorporated herein by reference.

Various approaches to DNA sequencing exist. The dideoxy chaintermination method serves as the basis for all currently availableautomated DNA sequencing machines. See, e.g., Sanger et al., Proc. Natl.Acad. Sci., 74: 5463-5467 (1977); Church et al., Science, 240: 185-188(1988); and Hunkapiller et al., Science, 254: 59-67 (1991)). Othermethods include the chemical degradation method, see, e.g., Maxam etal., Proc. Natl. Acad. Sci., 74: 560-564 (1977); whole-genome approachessee, e.g., Fleischmann et al., Science, 269, 496 (1995); expressedsequence tag sequencing see, e.g., Velculescu et al., Science, 270,(1995); array methods based on sequencing by hybridization, see, e.g.,Koster et al., Nature Biotechnology, 14, 1123 (1996); and singlemolecule sequencing (SMS), see, e.g., Jett et al., J. Biomol. Struct.Dyn. 7, 301 (1989), Schecker et al., Proc. SPIE-Int. Soc. Opt. Eng.2386, 4 (1995), and Hardin et al. U.S. patent application Ser. No.09/901,782, filed Jul. 9, 2001, incorporated herein by reference.

Fluorescent dyes can be used in a variety of these DNA sequencingtechniques. A fluorophore moiety or dye is a molecule capable ofgenerating a fluorescence signal. A quencher moiety is a moleculecapable of absorbing the energy of an excited fluorophore, therebyquenching the fluorescence signal that would otherwise be released fromthe excited fluorophore. In order for a quencher to quench an excitedfluorophore, the quencher moiety must be within a minimum quenchingdistance of the excited fluorophore moiety at some time prior to thefluorophore releasing the stored fluorescence energy.

Fluorophore-quencher pairs have been incorporated into oligonucleotideprobes in order to monitor biological events based on the fluorophoreand quencher being separated or brought within a minimum quenchingdistance of each other. For example, probes have been developed whereinthe intensity of the fluorescence increases due to the separation of thefluorophore-quencher pair. Probes have also been developed which losetheir fluorescence because the quencher is brought into proximity withthe fluorophore.

These fluorophore-quencher pairs have been used to monitor hybridizationassays and nucleic acid amplification reactions, especially polymerasechain reactions (PCR), by monitoring either the appearance ordisappearance of the fluorescence signal generated by the fluorophoremolecule.

The decreased fluorescence of a fluorophore moiety by collision ordirect interaction with a quencher is due mainly to a transfer of energyfrom the fluorophore in the excited state to the quencher. The extent ofquenching depends on the concentration of quencher and is described bythe Stern-Volmer relationship:

F ₀ /F=1+K _(sv) [Q]

wherein F₀ and F correspond to the fluorescence in the absence andpresence of quencher, respectively, and [Q] is the quencherconcentration. A plot of F₀/F versus [Q] yields a straight line with aslope corresponding to the Stern-Volmer constant, K_(sv). The foregoingequation takes into account the dynamic and collisional quenching whichis the dominant component of the quenching reaction. A linear S-V plotcan be obtained when the quenching is completely due to a dynamic (orcollisional) process or a static complex formation. A non-linear plotwill occur when both static and collisional quenching are occurringsimultaneously (see, A. M. Garcia, Methods in Enzymology, 207, 501-511(1992)).

In general, fluorophore moieties preferably have a high quantum yieldand a large extinction coefficient so that the dye can be used to detectsmall quantities of the component being detected. Fluorophore moietiespreferably have a large Stokes shift (i.e., the difference between thewavelength at which the dye has maximum absorbance and the wavelength atwhich the dye has maximum emission) so that the fluorescent emission isreadily distinguished from the light source used to excite the dye.

One class of fluorescent dyes which has been developed is the energytransfer fluorescent dyes. For instance, U.S. Pat. Nos. 5,800,996, and5,863,727, issued to Lee et al., disclose donor and acceptor energyfluorescent dyes and linkers useful for DNA sequencing, incorporatedtherein by reference. Other fluorophore-quencher pairs are disclosed inPCT Application Serial No. PCT/US99/29584, incorporated herein byreference. In energy transfer fluorescent dyes, the acceptor molecule isa fluorophore which is excited at the wavelength of light correspondingto the fluorescence emission the excited donor molecule. When excited,the donor dye transmits its energy to the acceptor dye.

Therefore, emission from the donor is partially or totally quenched dueto partial or total energy transfer from the excited donor to theacceptor dye, resulting in the excitation of the latter for emission atits characteristic wavelength (i.e., a wavelength different from that ofthe donor dye which may represent a different color if the emissions arein the visible portion of the spectrum). The advantage of this mechanismis twofold; the emission from the acceptor dye is more intense than thatfrom the donor dye alone when the acceptor has a higher fluorescencequantum yield than the donor (see, Li et al., Bioconjugate Chem., 10:242-245, (1999)) and attachment of acceptor dyes with differing emissionspectra allows differentiation among molecules by fluorescence using asingle excitation wavelength.

Nucleotide triphosphates having a fluorophore moiety attached to theγ-phosphate are of interest as this modification still allows themodified NTPs to be enzyme substrates. For instance, Felicia et al.,describe the synthesis and spectral properties of a “always-on”fluorescent ATP analog, adenosine-5′-triphosphoroyl-(5-sulfonicacid)naphthyl ethylamindate (γ-1,5-EDANS) ATP. Yarbrough et al. 1978,JBC. The analog is a good substrate for E. coli RNA polymerase and canbe used to initiate the RNA chain. The ATP analog is incorporated intothe RNA synthesized and is a good probe for studies ofnucleotide-protein interactions, active site mapping and otherATP-utilizing biological systems. See, e.g., Felicia et al., Arch.Biochem Biophys., 246: 564-571 (1986).

In addition, Sato et al., disclose a homogeneous enzyme assay that usesa fluorophore moiety (bimane) attached to the γ-phosphate group of thenucleotide and a quencher moiety attached to the 5-position of uracil.The quencher moiety is in the form of a halogen, bound to the C-5position of the pyrimidine. The quenching that is effected by thiscombination is eliminated by cleavage of the phosphate bond by thephosphodiesterase enzyme. The halogen quencher used in the assay is veryinefficient producing only about a two fold decrease in fluorescentefficiency.

Template-Mediated Primer Extension Reaction

In a template-mediated primer extension reaction, an oligonucleotideprimer having homology to a single-stranded template nucleic acid iscaused to anneal to a template nucleic acid, the annealed mixture isthen provided with a DNA polymerase in the presence of nucleosidetriphosphates under conditions in which the DNA polymerase extends theprimer to form a complementary strand to the template nucleic acid. In aSanger-type DNA sequencing reaction, the primer is extended in thepresence of a chain-terminating agent, e.g., a dideoxynucleosidetriphosphate, to cause base-specific termination of the primer extension(Sanger). In a polymerase chain reaction, two primers are provided, eachhaving homology to opposite strands of a double-stranded DNA molecule.After the primers are extended, they are separated from their templates,and additional primers caused to anneal to the templates and theextended primers. The additional primers are then extended. The steps ofseparating, annealing, and extending are repeated in order togeometrically amplify the number of copies of the template nucleic acid(Saiki).

In both DNA sequencing and PCR, it is critically important that theprimer extension product accurately replicate the nucleotide sequence ofthe template nucleic acid. However, under certain conditions, peak“dropout” has been observed wherein certain nucleotides are notrepresented in the primer extension product. This problem is believed tobe caused by pyrophosphorolysis of the primer extension product by areverse nucleotide addition reaction promoted by the accumulation ofpyrophosphates in the reaction mixture. See Mullis; Tabor 1990; Tabor1996.

Pyrophosphate Effects on Nucleic Acid Synthesis and/or Sequencing

It has been recognized that pyrophosphorolysis, where an oligonucleotideis reduced in length, is detrimental to primer extension reactions. Thepyrophosphorolysis is caused by the availability of pyrophosphate. Forexample, PCR is inhibited by the addition of pyrophosphate even at verylow concentrations. According to U.S. Pat. No. 5,498,523, thispyrophosphorolysis can be prevented by providing an agent, for example,a pyrophosphatase, capable of removing pyrophosphate. Addition ofpyrophosphatase to a PCR greatly enhances the progress of the reactionand provides superior results compared to the reaction without apyrophosphatase. See U.S. Pat. No. 4,800,159, incorporated herein byreference.

Similarly, the addition of a pyrophosphatase to a sequencing reactionprovides more uniformity in intensities of bands formed in apolyacrylamide gel used to identify products of the sequencing reaction.This uniformity is due to prevention of degradation of specific DNAproducts by pyrophosphorolysis. See also, Tabor, S. and Richardson, C.C., J. Biol. Chem. 265:8322 (1990) and U.S. Pat. No. 4,962,020,incorporated herein by reference.

Each product or band in a dideoxy sequencing experiment is apolynucleotide complementary to the template and terminated at the 3′end in a base-specific manner with a dideoxynucleotide. The dideoxystabilizes the product, preventing further polymerization of thepolynucleotide. However, in certain regions of the template, the bands,especially after prolonged reaction, will reduce in intensity orcompletely disappear (“drop-out” bands). In certain sequence contexts,the PPi contained within the enzyme is thought to remain there for anextended period of time. A drop-out may not be readily detected by theoperator, leading to errors in the interpretation of the data either bya human or computer-driven analyzer. Since this phenomenon is stimulatedby inorganic pyrophosphate, the effect is presumably due topyrophosphorolysis (reverse polymerization), not 3′-exonucleolyticactivity. It is hypothesized that DNA polymerase idling at the end ofthese terminated products and in the presence of sufficientpyrophosphate will remove the dideoxynucleotide, then extend from thenow free 3′-hydroxyl end to another dideoxy termination. In effect, thebands are converted to longer polynucleotides bands. Removal ofpyrophosphate as it is generated in the polymerization reactioneliminates this problem.

Sequencing by Direct Detection of Released Tagged Pyrophosphate

Researchers have used a series of enzyme reactions coupled topyrophosphate generation to measure DNA polymerase activity. In thefirst (P. Nyren, Anal. Biochem. 167:235 (1987)), Nyren used ATP: sulfateadenylyltransferase to convert pyrophosphate and adenosine5′-phosphosulfate to ATP and sulfate ion. The ATP was used to make lightwith luciferase. In the second (J. C. Johnson et al., Anal. Biochem.26:137 (1968)), the researchers reacted the pyrophosphate withUDP-glucose in the presence of UTP: glucose-1-phosphateuridylyltransferase to produce UTP and glucose-1-phosphate. In two moresteps, polymerase activity was measured spectrophotometrically by theconversion of NADP to NADPH. While these articles describe the use ofATP: sulfate adenylyltransferase and UTP: glucose-1-phosphateuridylyltransferase in measuring DNA polymerase activity, they do notdescribe their use to prevent or inhibit pyrophosphorolysis in nucleicacid synthesis reactions.

DNA sequencing is an essential tool in molecular genetic analysis. Theability to determine DNA nucleotide sequences has become increasinglyimportant as efforts have commenced to determine the sequences of thelarge genomes of humans and other higher organisms.

The two most commonly used methods for DNA sequencing are the enzymaticchain-termination method of Sanger and the chemical cleavage techniqueof Maxam and Gilbert.

Both methods rely on gel electrophoresis to resolve, according to theirsize, DNA fragments produced from a larger DNA segment. Since theelectrophoresis step as well as the subsequent detection of theseparated DNA fragments are cumbersome procedures, a great effort hasbeen made to automate these steps. However, despite the fact thatautomated electrophoresis units are commercially available,electrophoresis is not well suited for large-scale genome projects orclinical sequencing where relatively cost-effective units with highthroughput are needed. Thus, the need for nonelectrophoretic methods forsequencing is great and several alternative strategies have beendescribed, such as scanning tunnel electron microscopy (Driscoll et al.1990, Nature, 346, 294-296), sequencing by hybridization (Bains et al.,1988, J. Theo. Biol. 135, 308-307) and single molecule detection (Jeffet al., 1989, Biomol. Struct. Dynamics, 7, 301-306), to overcome thedisadvantages of electrophoresis.

Techniques enabling the rapid detection of a single DNA base change arealso important tools for genetic analysis. In many cases detection of asingle base or a few bases would be a great help in genetic analysissince several genetic diseases and certain cancers are related to minormutations. A mini-sequencing protocol based on a solid phase principlewas described (Hultman, et al., 1988, Nucl. Acid. Res., 17, 4937-4946;Syvanen et al., 1990, Genomics, 8, 684-692). The incorporation of aradio labeled nucleotide was measured and used for analysis of thethree-allelic polymorphism of the human apolipoprotein E gene. However,radioactive methods are not well suited for routine clinicalapplications and hence the development of a simple non-radioactivemethod for rapid DNA sequence analysis has also been of interest.

Methods of sequencing based on the concept of detecting inorganicpyrophosphate (PPi) which is released during a polymerase reaction havebeen described (WO 93/23564 and WO 89/09283). As each nucleotide isadded to a growing nucleic acid strand during a polymerase reaction, apyrophosphate molecule is released. It has been found that pyrophosphatereleased under these conditions can be detected enzymically e.g. by thegeneration of light in the luciferase-luciferin reaction. Such methodsenable a base to be identified in a target position and DNA to besequenced simply and rapidly whilst avoiding the need forelectrophoresis and the use of harmful radio labels. See for exampleU.S. Pat. No. 5,498,523, incorporated herein by reference.

However, the PPi-based sequencing methods mentioned above are notwithout drawbacks. The template must be washed thoroughly between eachnucleotide addition to remove all non-incorporated deoxynucleotides.This makes it difficult to sequence a template which is not bound to asolid support. In addition new enzymes must be added with each additionof deoxynucleotide.

Thus, there is a need for improved methods of sequencing which allowrapid detection, have increase fidelity and provision of sequenceinformation and which are simple and quick to perform, lendingthemselves readily to automation.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies of the prior art andprovides a nucleotide polymerization using nucleotides having amolecular and/or atomic tag bonded to or associated with the nucleotideor nucleoside to alter fidelity of nucleotide incorporation. In apreferred embodiment, the tag is bonded to or associated with a portionof the nucleotide that is released after nucleotide incorporation in agrowing polymer chain. Preferably, the released portion is thepyrophosphate moiety including the β and γ phosphate groups.

When a pyrophosphate group having a molecular and/or atomic tag bondedto or associated therewith is released from the nucleoside triphosphateupon incorporation in a growing polymer chain, the tagged pyrophosphategroup does not (significantly) stimulate pyrophosphorolysis.

The present invention also provides a method for preventing reversepolymerization or depolymerization of polymer formed usingsingle-molecule sequencing methods as set forth in U.S. Prov. Pat.Appln. Ser. No. 60/216,594, filed Jul. 7, 2000 and U.S. patentapplication Ser. No. 09/901,782, filed Jul. 9, 2001, incorporated hereinby reference.

The present invention further provides a method for improvingincorporation fidelity by adding a modified PP_(i) to a nucleosidepolymerization medium in an amount sufficient to improve incorporationfidelity and/or to inhibit of pyrophosphorolysis of formed products.Preferred modified pyrophosphates include pyrophosphates bearing a groupon one or both phosphate moieties that reduce, inhibit or preventpyrophosphorolysis or pyrophosphates produced from NTPs or dNTPs havinga group on the β and/or γ phosphate moiety.

The present invention provides a heterogeneous assay for detecting baseincorporation and pyrophosphate cleavage. The assay utilizes labeledNTPs or dNTPs, a target nucleic acid, a primer nucleic acid and apolymerase. The assay includes the steps of flowing the labelednucleotide triphosphate (NTP, dNTP, etc.) having a molecular and/oratomic tag bonded to or associated with the β- and/or γ-phosphate pastan immobilized component selected from the group consisting of thepolymerase, the primer and the target nucleic acid. Next, theappropriate labeled NTP or dNTP is incorporated on the primer strandhybridized to the target nucleic acid using the polymerase and resultsin the release of a tagged pyrophosphate from the dNTP. Theincorporation event or the release event can be detected either bymeasuring a detectable property of the NTP or dNTP upon binding and/orduring incorporation or by measuring a detectable property of thereleased pyrophosphate. The detectable property can be a propertyinherent in the molecular or atomic tags or produced as a result of theinteraction between the molecular or atomic tag on the phosphates of thelabel NTP or released pyrophosphate and other tags bonded to orassociated with the polymerase, the matrix or mobile or immobilecomponents in the media.

The present invention also provides a polymerase immobilized on a solidsupport and a labeled nucleotide triphosphate selected from the groupconsisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, UTP andmixtures thereof, where the tags are molecular and the molecules arefluorophores and the detectable property is fluorescent light emissionor quenching. The detection of the fluorescent light is preferablyaccomplished using single molecule detection such as a charge coupledevice (CCD) camera or intensified CCD camera systems or the like.

The present invention provides kits and integrated systems forpracticing the assays described herein. In certain aspects, the presentinvention provides a kit for assaying pyrophosphate cleavage,comprising: (a) a plurality of nucleotides triphosphates each having aγ-phosphate with a distinguishing fluorophore moiety attached theretoand each having a quencher moiety sufficiently proximal to thedistinguishing fluorophore moiety to prevent fluorescence of thedistinguishing fluorophore moiety; wherein the distinguishingfluorophore moiety exists quenched with at least about a 5 foldquenching efficiency when the γ-phosphate is attached to each of theplurality of dNTP moieties and each is unquenched when the γ-phosphateis detached from each of the plurality of dNTP moieties; and (b) apolymerase. Preferably, the polymerase is immobilized on a solidsupport.

The present invention provides a primer extension method in which theextent of pyrophosphorolysis of a primer extension product is reduced,and solutions and kits useful for practicing the method.

The present invention provides a primer extension method wherein “peakdrop-out” is reduced and the fidelity of template-sequence reproductionis maximized.

The present invention provides an improved method for performing aprimer extension reaction including the steps of annealing anoligonucleotide primer to a portion of a template nucleic acid therebyforming a primer template hybrid; adding primer-extension reagentsincluding a NTP or dNTP having a β- and/or γ-phosphate moiety having amolecular and/or atomic tag bonded to or associated with the β- and/orγ-phosphate moiety to the primer-template hybrid for extending theprimer; and optionally adding a co-substrate-enzyme pair to theprimer-template hybrid for conducting a pyrophosphate-utilizingreaction, where the tagged, released pyrophosphate reduces the amount ofpyrophosphorolysis in the reaction. One should recognize that therelease PP_(i) is a modified PP_(i) and acts to inhibit deleteriousinterference untagged PP_(i) has on nucleotide polymerization.

The present invention provides a method of inhibiting or preventingpyrophosphorolysis during synthesis of a nucleic acid molecule, saidmethod comprising: (a) combining one or more nucleotides having amolecular and/or atomic tag bonded to or associated with a β- and/orγ-phosphate moiety of the nucleoside and a nucleic acid template; (b)incubating the one or more nucleotides and nucleic acid template, underconditions sufficient to form a second nucleic acid moleculecomplementary to all or a portion of the nucleic acid template.

The method of the invention more specifically relates to a method ofinhibiting or preventing pyrophosphorolysis, said method comprising: (a)combining a primer with a nucleic acid template under conditionssufficient to form a hybridized product; and (b) incubating saidhybridized product in the presence of (i) one or more nucleotides havinga molecular and/or atomic tag bonded to or associated with a β- and/orγ-phosphate moiety of the nucleoside, and (ii) a polymerase, and (iii)optionally an enzyme selected from the group consisting of apentosyltransferase, a phosphotransferase with alcohol group asacceptor, a nucleotidyltransferase, and a carboxy-lyase under conditionssufficient to synthesize a second nucleic acid molecule complementary toall or a portion of said nucleic acid template.

Specifically, the method of the present invention relates to inhibitionof pyrophosphorolysis in the synthesis of DNA and RNA molecules usingthe appropriate nucleotides having a molecular and/or atomic tag bondedto or associated with a β- and/or γ-phosphate moiety of the nucleosideand polymerases (dNTPs/rNTPs and DNA polymerase/RNA polymerase).

The present invention provides a primer extension reaction to preventthe inhibition of nucleic acid synthesis during amplification and toprevent band drop out in sequencing reactions. Thus, the method toprevent inhibition of nucleic acid synthesis during amplification of adouble stranded nucleic acid molecule comprises: (a) providing a firstand second primer, wherein said first primer is complementary to asequence at or near the 3′ termini of the first strand of said nucleicacid molecule and said second primer is complementary to a sequence ator near the 3′ termini of the second strand of said nucleic acidmolecule; (b) hybridizing said first primer to said first strand andsaid second primer to said second strand in the presence of (i) apolymerase, and (ii) optionally an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with analcohol group as an acceptor, a nucleotidyltransferase and acarboxy-lyase under conditions such that a third nucleic acid moleculecomplementary to said first strand and a fourth nucleic acid moleculecomplementary to said second strand are synthesized from nucleosideshaving a molecular and/or atomic tag bonded to or associated with a β-and/or γ-phosphate moiety of the nucleoside; (c) denaturing said firstand third strand and said second and fourth strand; and (d) repeatingsteps (a) to (c) one or more times. Again, the PP_(i) released in thenucleotide polymerization of this invention do not cause the deleteriouseffects that nascent PP_(i) can cause, therefore, the need toenzymatically degrade PP_(i) is only for optional protection.

The present invention also provides a method of sequencing a DNAmolecule comprising: (a) combining a primer with a first DNA moleculeunder conditions sufficient to form a hybridized product; (b) contactingsaid hybridized product with nucleotides having a molecular and/oratomic tag bonded to or associated with a β- and/or γ-phosphate moietyof the nucleoside, a DNA polymerase, optionally an enzyme selected fromthe group consisting of a pentosyltransferase, a phosphotransferase withan alcohol group as acceptor, a nucleotidyltransferase and acarboxy-lyase; and a terminator nucleotide to give a reaction mixture;(c) incubating the reaction mixture under conditions sufficient tosynthesize a population of DNA molecules complementary to said first DNAmolecule, wherein said synthesized DNA molecules are shorter in lengththan said first DNA molecule and wherein said synthesized DNA moleculescomprise a terminator nucleotide at their 3′ termini; and (d) separatingsaid synthesized DNA molecules by size so that at least a part of thenucleotide sequence of said first DNA molecule can be determined.

In addition to reducing band drop out, which is believed to result froma ddNTP being added and then being release due to reattaching releasepyrophosphate followed by standard extension, thereby, producing underrepresentation of that position in the DNA sequence data, the use of β-and/or γ-phosphate modified nucleotides will result in improvedsequencing using traditional fluorescent sequencing reaction due to adecrease in background and/or reduction in band spreading. The firstimprovement would result from using β- and/or γ-phosphate modifieddideoxynucleotides, which are incorporated at improved accuracy (lessincorporation of incorrect ddNTP, reducing background signal). While thesecond improvement would result from using β- and/or γ-phosphatemodified nucleotides to produce identical (or substantially identical)DNA polymers instead of the population of molecules that result frominaccurate incorporation of dNTPs. Thus, the traditional fluorescentsequencing reaction can undergo a two stage improvement by using β-and/or γ-phosphate modified nucleotides and β- and/or γ-phosphatemodified dideoxy nucleotides.

The present invention provides a novel modified PP_(i)-based sequencingmethod for sequencing reactions, where the method can be performedwithout intermediate washing steps, enabling the procedure to be carriedout simply and rapidly, for example in a single micro titre plate.Moreover, the method can be performed with immobilized DNA in solutionor on a support or with mobile DNA and immobilized polymerase insolution or on a support. Furthermore, the method can be readily adaptedto permit the sequencing reactions to be continuously monitored inreal-time, with a signal being generated and detected, as eachnucleotide is incorporated.

The present invention provides a method of identifying a base at atarget position in a sample DNA sequence wherein an extension primer,which hybridizes to the sample DNA immediately adjacent to the targetposition is provided and the sample DNA and extension primer aresubjected to a polymerase reaction in the presence of a deoxynucleotidehaving a molecular and/or atomic tag bonded to or associated with a β-and/or γ-phosphate moiety of the nucleoside or dideoxynucleotide havinga molecular and/or atomic tag bonded to or associated with a β- and/orγ-phosphate moiety of the nucleoside whereby the tagged deoxynucleotideor tagged dideoxynucleotide will only become incorporated and releasetagged pyrophosphate (tPPi) if it is complementary to the base in thetarget position, any incorporation and/or release of tPPi may bedetected via any detection method capable of identifying a detectableproperty of the tagged deoxynucleotide, tagged dideoxynucleotide ortagged pyrophosphate, different tagged deoxynucleotides or taggeddideoxynucleotides being added either to separate aliquots ofsample-primer mixture or successively to the same sample-primer mixtureand subjected to the polymerase reaction to indicate which taggeddeoxynucleotide or tagged dideoxynucleotide is incorporated, optionallycharacterised in that, a nucleotide-degrading enzyme is included duringthe polymerase reaction step, such that unincorporated nucleotides areeliminated.

The present invention is also ideally suited for single nucleotideextensions reactions because the tagged PP_(i) released duringincorporation does not cause the deleterious effects associated with therelease of nascent PP_(i), and where the fidelity of the taggednucleotide incorporation in improved.

The invention also provides a kit for carrying out nucleic acidsyntheses with improved fidelity comprising a container including apolymerizing compartment comprising a nucleic acid polymerizing agent, amonomer compartment comprising nucleotide monomers for the polymerizingagent and a fidelity enhancing agent compartment comprising a fidelityenhancing agent, where the fidelity enhancing agent comprises atagged-phosphate, tagged-pyrophosphate or tagged-polyphosphate orderivatives thereof.

The invention also provides a kit for carrying out nucleic acidsyntheses with improved fidelity comprising a container including apolymerizing compartment comprising a nucleic acid polymerizing agentand a monomer compartment comprising nucleotide monomers for thepolymerizing agent, where the monomers comprise dNTPs, ddNTPs, β- and/orγ-phosphate modified nucleotides, β- and/or γ-phosphate modified dideoxynucleotides or mixtures or combinations thereof.

The invention also provides a kit for carrying out nucleic acidsyntheses with improved fidelity comprising a container including apolymerizing compartment comprising a nucleic acid polymerizing agentand monomer compartments, each compartment comprising a nucleotidemonomer for the polymerizing agent, where the monomers comprise dNTPs,ddNTPs, β- and/or γ-phosphate tagged dNTPs, β- and/or γ-phosphate taggedddNTPs or mixtures or combinations thereof.

DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdetailed description together with the appended illustrative drawings inwhich like elements are numbered the same:

FIG. 1 depicts the incorporation of ANS-γ-phosphate dATP using Taqpolymerase and a primer;

FIG. 2 depicts the synthesis of extended DNA polymers using all fourANS-γ-phosphated tagged dNTPs and the Taq polymerase;

FIG. 3 depicts the synthesis of long DNA polymers using all fourANS-γ-phosphated tagged dNTPs and the Taq polymerase;

FIG. 4 depicts the use of γ-modified nucleotides with the Klenowfragment from E. coli DNA polymerase to form DNA polymer;

FIG. 5 depicts the use of γ-modified nucleotides with the Pfu DNApolymerase that shows this polymerase does not efficiently useγ-modified nucleotides;

FIG. 6 depicts the use of γ-modified nucleotides using HIV-1 reversetranscriptase to efficiently form DNA polymers;

FIG. 7 depicts the experimental results for native T7 DNA polymerase andSequenase;

FIG. 8 depicts the effect of elevated temperature on ANS-tagged dATPsand ANS-tagged dATPs;

FIG. 9 depicts the effect of elevated temperature on ANS-tagged dCTPsand ANS-tagged dGTPs;

FIG. 10 depicts the effect of temperature and time on the ability of TaqDNA Polymerase to produce extended DNA products from primer/templateduplexes;

FIG. 11 depicts the addition of an ANS-γ-tag to natural dNTPs affectsthe terminal transferase activity of commercially available Taq DNAPolymerase;

FIG. 12 depicts a summary of extension results for various polymeraseincorporating ANS-tagged dNTPs;

FIG. 13 depicts data from time course experiments demonstrating similarincorporation of natural and γ-phosphate modified nucleotide using HIVreverse transcriptase; and

FIG. 14A: Representative gels demonstrating the results obtained insingle nucleotide extension assays using the Bot-C template.Incorporation of matched (dGTP & ANS-dGTP, above) and mismatched (dTTP &ANS-dTTP, below) nucleotides are shown.

FIG. 14B: Graphic presentation of the fidelity improvements afforded byANS addition to the γ-phosphate of each dNTP. The increase in percentextension of the natural nucleotide relative to the ANS-taggednucleotide is indicated above the natural nucleotide.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The term “heterogeneous” assay as used herein refers to an assay methodwherein at least one of the reactants in the assay mixture is attachedto a solid phase, such as a solid support.

The term “oligonucleotide” as used herein includes linear oligomers ofnucleotides or analogs thereof, including deoxyribonucleosides,ribonucleosides, and the like. Usually, oligonucleotides range in sizefrom a few monomeric units, e. g. 3-4, to several hundreds of monomericunits. Whenever an oligonucleotide is represented by a sequence ofletters, such as “ATGCCTG” SEQ. ID 1, it will be understood that thenucleotides are in 5′-3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes thymine, unless otherwise noted.

The term “nucleoside” as used herein refers to a compound consisting ofa purine, deazapurine, or pyrimidine nucleoside base, e. g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e. g., as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992) and further include,but are not limited to, synthetic nucleosides having modified basemoieties and/or modified sugar moieties, e. g. described generally byScheit, Nucleotide Analogs (John Wiley, N.Y., 1980). Suitable NTPsinclude both naturally occurring and synthetic nucleotide triphosphates,and are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, TTP, dTTP, ITP,dITP, UTP and dUTP. Preferably, the nucleotide triphosphates used in themethods of the present invention are selected from the group of dATP,dCTP, dGTP, dTTP, dUTP and mixtures thereof.

The term “nucleotide” as used herein refers to a phosphate ester of anucleoside, e.g., mono, di and triphosphate esters, wherein the mostcommon site of esterification is the hydroxyl group attached to the C-5position of the pentose and includes deoxyribonucleoside triphosphatessuch as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof suchas their dideoxy derivatives: ddATP, ddCTP, ddITP, ddUTP, ddGTP, ddTTP.Such derivatives include, for example [aS]dATP, 7-deaza-dGTP and7-deaza-dATP. The term “nucleotide” as used herein also refers toribonucleoside triphosphates (NTPs) and their derivatives. Illustratedexamples of ribonucleoside triphosphates include, but are not limitedto, ATP, CTP, GTP, ITP and UTP.

The term “primer” refers to a linear oligonucleotide which specificallyanneals to a unique polynucleotide sequence and allows for amplificationof that unique polynucleotide sequence or to a nucleic acid, e.g.,synthetic oligonucleotide, which is capable of annealing to acomplementary template nucleic acid and serving as a point of initiationfor template-directed nucleic acid synthesis. Typically, a primer willinclude a free hydroxyl group at the 3′-end.

The phrase “sequence determination” or “determining a nucleotidesequence” in reference to polynucleotides includes determination ofpartial as well as full sequence information of the polynucleotide. Thatis, the term includes sequence comparisons, fingerprinting, and likelevels of information about a target polynucleotide, or oligonucleotide,as well as the express identification and ordering of nucleotides,usually each nucleotide, in a target polynucleotide. The term alsoincludes the determination of the identification, ordering, andlocations of one, two, or three of the four types of nucleotides withina target polynucleotide.

The term “solid-support” refers to a material in the solid-phase thatinteracts with reagents in the liquid phase by heterogeneous reactions.Solid-supports can be derivatized with proteins such as enzymes,peptides, oligonucleotides and polynucleotides by covalent ornon-covalent bonding through one or more attachment sites, thereby“immobilizing” the protein or nucleic acid to the solid-support.

The phrase “target nucleic acid” or “target polynucleotide” refers to anucleic acid or polynucleotide whose sequence identity or ordering orlocation of nucleosides is to be determined using methods describedherein.

The term “primer-extension reagent” means a reagent including componentsnecessary to effect the enzymatic template-mediated extension of aprimer. Primer extension reagents include: (i) a polymerase enzyme,e.g., a thermostable polymerase enzyme such as Taq DNA polymerase, andthe like; (ii) a buffer to stabilize pH; (iii) deoxynucleotidetriphosphates, e.g., deoxyguanosine 5′-triphosphate,7-deazadeoxyguanosine 5′-triphosphate, deoxyadenosine 5′-triphosphate,deoxythymidine 5′-triphosphate, deoxycytidine 5′-triphosphate; and,optionally in the case of a Sanger-type DNA sequencing reaction, (iv)dideoxynucleotide triphosphates, e.g., dideoxyguanosine 5′triphosphate,7-deazadideoxyguanosine 5′-triphosphate, dideoxyadenosine5′-triphosphate, dideoxythymidine 5′-triphosphate, dideoxycytidine5′-triphosphate, and the like.

As used herein, the term “pyrophosphate” refers to two phosphatemolecules bound together by an ester linkage, e.g., the structure⁻²O³P—O—PO₃ ⁻².

The term “nucleotide-degrading enzyme” as used herein includes allenzymes capable of non-specifically degrading nucleotides, including atleast nucleoside triphosphates (NTPs), but optionally also di- andmonophosphates, and any mixture or combination of such enzymes, providedthat a nucleoside triphosphatase or other NTP degrading activity ispresent. Although nucleotide-degrading enzymes having a phosphataseactivity may conveniently be used according to the invention, any enzymehaving any nucleotide or nucleoside degrading activity may be used,e.g., enzymes which cleave nucleotides at positions other than at thephosphate group, for example at the base or sugar residues. Thus, anucleoside triphosphate degrading enzyme is essential for the invention.

The term “atomic tag” means an atom or ion of an atom that when attachedto a nucleotide increase the fidelity of a nucleotide polymerizing agentsuch as a polymerase at the atom tagged nucleotide is incorporated intoa nucleotide sequence.

The term “molecular tag” means an atom or ion of an atom that whenattached to a nucleotide increase the fidelity of a nucleotidepolymerizing agent such as a polymerase at the atom tagged nucleotide isincorporated into a nucleotide sequence.

The term “polymerizing agent” means any naturally occurring or syntheticagent capable of polymerizing nucleotides to produce polynucleotide,including polymerases, reverse transcriptases, or the related naturallyoccurring nucleotide polymerizing systems. The term polymerizing agentalso includes variants of naturally occurring polymerases or reversetranscriptases where one or more amino acids have been added to, removedfrom or replaced in the nature amino acid sequence. Thus, the termcovers all known and to be constructed systems capable of formingoligomers or polymers of nucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that nucleotide monomers or analogs thereofbearing an atomic and/or molecular tag on a site of the molecule canincrease the fidelity of nucleotide polymerization for nucleotidepolymerization agents that can incorporated the modified monomers. Thisincrease in fidelity is useful for improving nucleic acid sequencingdeterminations using any of the standard sequencing reactions such asPCR, rolling circle or the like. Additionally, these modified monomersmay allows the construction of drugs for animal or human use that wouldincrease the fidelity of viral disease replication in vivo decreasingmutagensis allowing the immune system to recognize the virus. Such amedication may be of particular benefit for virus such as the HIV virusthat causes AIDS.

Mutation of amino acids within the polymerase is the classic approach tounderstand enzyme action and/or modulate enzyme fidelity (Yang S andChatterjee D K. (1999) PCT WO9910366; Wainberg M A, Drosopoulos W C,Salomon H, Hsu M, Borkow G, Parniak M, Gu Z, Song Q, Manne J, Islam S,Castriota G, Prasad V R. (1996) Science 271:1282-5; Drosopoulos W C,Rezende L F, Wainberg M A, Prasad V R. (1998) J Mol Med 76:604-12; LewisD A, Bebenek K, Beard W A, Wilson S H, Kunkel T A. (1999) J Biol Chem274:32924-30; Kim B, Ayran J C, Sagar S G, Adman E T, Fuller S M, Tran NH, Horrigan J. (1999) J Biol Chem 274:27666-73. In stark distinction tothis classical approach, the inventors have found a novel approach inthat fidelity is improved by manipulating the substrate.

Invention Scope

The present invention relates to a composition comprising a nucleotidesincluding deoxyribonucleotide, dideoxynucleotide, or ribonucleotideincluding a molecular and/or atomic tag on a β and/or γ phosphate groupand/or a base moiety, where the tag alters fidelity of baseincorporation.

The present invention relates to a method comprising the step of addinga composition comprising a nucleotides including deoxyribonucleotide,dideoxynucleotide, or ribonucleotide including a molecular and/or atomictag on a β and/or γ phosphate group and/or a base moiety, where the tagalters fidelity of base incorporation to a nucleotide polymerizationmedium comprising a nucleotide polymerase.

The present invention relates to a composition comprising a nucleotidesincluding deoxyribonucleotide, dideoxynucleotide, or ribonucleotideincluding a molecular and/or atomic tag on a β phosphate group and/or abase moiety, where the tag alters fidelity of base incorporation.

The present invention relates to a method comprising the step of addinga composition comprising a nucleotides including deoxyribonucleotide,dideoxynucleotide, or ribonucleotide including a molecular and/or atomictag on a β phosphate group and/or a base moiety, where the tag altersfidelity of base incorporation to a nucleotide polymerization mediumcomprising a nucleotide polymerase.

The present invention relates to a composition comprising a nucleotidesincluding deoxyribonucleotide, dideoxynucleotide, or ribonucleotideincluding a molecular and/or atomic tag on a γ phosphate group and/or abase moiety, where the tag alters fidelity of base incorporation.

The present invention relates to a method comprising the step of addinga composition comprising a nucleotides including deoxyribonucleotide,dideoxynucleotide, or ribonucleotide including a molecular and/or atomictag on a γ phosphate group and/or a base moiety, where the tag altersfidelity of base incorporation to a nucleotide polymerization mediumcomprising a nucleotide polymerase.

The present invention relates to a method comprising the step of addinga nucleotides including deoxyribonucleotide, dideoxynucleotide, orribonucleotide including a molecular and/or atomic tag on a β and/or γphosphate group to an assay involving a polymerase and/or a base moiety,where the tag alters fidelity of base incorporation and the assay isselected from the group consisting of genotyping for in vitroreproductive methods (human and other organisms); single nucleotidepolymorphism (SNP) detection; DNA sequencing; RNA sequencing; singlenucleotide extension assays; amplified DNA product assays; rollingcircle product assays; PCR product assays; allele-specific primerextension assays; single-molecule arrays (DNA, RNA, protein) assays;drug toxicity evaluation assays; or the like. The method can be used toextend a nucleic acid molecule by any number of bases depending on thepolymerizing reaction selected. Thus, the molecule can be extended by asingle nucleotide up to many thousands of nucleotide to or hundred ofthousands of bases.

The present invention relates to a method for making blunt-endedfragments comprising the steps of amplifying a DNA fragment in thepresence of a nucleotides including deoxyribonucleotide,dideoxynucleotide, or ribonucleotide including a molecular and/or atomictag on a γ phosphate group and/or a base moiety, where the tag altersfidelity of base incorporation and decreases or eliminates non-templatedaddition of a base to the 3′ end of the DNA fragment being amplified.Preferably, the amplifying step is a PCR amplification step.

The present invention relates to a composition comprising apyrophosphorolysis inhibitors selected from the group consisting ofcompounds of the following general formulas or mixtures or combinationsthereof:

Z—OPO₂O—Z′  (a)

Z—PO₂O—Z′  (b)

Z—OPO₂—Z′  (c)

Z—PO₂—Z′  (d)

Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)

Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)

Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)

Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h)

where Z or Z′ is a hydrogen atom or a thermally stable substituentcomprising primarily one or more atoms selected from the group carbon,nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atomsto satisfy valence requirements, E and E′ are an oxygen atom or athermally stable substituent comprising primarily one or more atomsselected from the group carbon, nitrogen, oxygen, sulfur and phosphoruswith sufficient hydrogen atoms to satisfy valence requirements and n isan integer having a value between 0 and about 5.

The present invention relates to a method comprising the step ofpolymerizing a nucleic acid sequence in the presence of a compositioncomprising a pyrophosphorolysis inhibitors selected from the groupconsisting of compounds of the following general formulas or mixtures orcombinations thereof:

Z—OPO₂O—Z′  (a)

Z—PO₂O—Z′  (b)

Z—OPO₂—Z′  (c)

Z—PO₂—Z′  (d)

Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)

Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)

Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)

Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h)

where Z or Z′ is a hydrogen atom or a thermally stable substituentcomprising primarily one or more atoms selected from the group carbon,nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atomsto satisfy valence requirements, E and E′ are an oxygen atom or athermally stable substituent comprising primarily one or more atomsselected from the group carbon, nitrogen, oxygen, sulfur and phosphoruswith sufficient hydrogen atoms to satisfy valence requirements and n isan integer having a value between 0 and about 5.

The present invention relates to a heterogeneous assay method fordetecting pyrophosphate cleavage, the components of the assay comprisinga labeled NTP, a target nucleic acid, a primer nucleic acid and apolymerase, said method comprising: (a) flowing said tagged nucleotidetriphosphate (NTP), where a β and/or γ phosphate group and/or a basemoiety of the NTP includes an atomic and/or molecular tag having adetectable property attached thereto or associated therewith; (b)incorporating said NTP on a primer strand hybridized to said targetnucleic acid using said polymerase and releasing said γ-phosphate withsaid fluorophore moiety attached thereto; and (c) detecting saidfluorescent moiety thereby detecting pyrophosphate cleavage. In onepreferred assay, the nucleotide triphosphate (NTP) is a member selectedfrom the group consisting of deoxyadenosine triphosphate, deoxycytosinetriphosphate, deoxyguanosine triphosphate and deoxythymidinetriphosphate. In another preferred assay, the nucleotide triphosphate(NTP) is a member selected from the group consisting of adenosinetriphosphate, cytosine triphosphate, guanosine triphosphate and uridinetriphosphate. In another preferred assay, the tags are a fluorescentspecies which is detected based upon a change in either intensitymeasurement or fluorescent lifetime measurement. In another preferredassay, the nucleotide triphosphate (NTP) is a plurality of nucleotidetriphosphates (NTPs). In another preferred assay, each of said pluralityof nucleotide triphosphates (NTPs) has an indicator of identityassociated with the tag. In another preferred assay, the polymerase is amember selected from the group consisting of a DNA polymerase, a DNAdependent RNA polymerase and a reverse transcriptase, particularly,where the polymerase is a DNA polymerase, especially, where thepolymerase is immobilized on a solid support. In another preferredassay, the polymerase is supported on a solid support that is a memberselected from the group consisting of controlled pore glass, a glassplate, polystyrene, an avidin coated polystyrene bead, cellulose, nylon,acrylamide gel and activated dextran.

The present invention relates to a nucleotide triphosphate (NTP) probecomprising a NTP including an atomic and/or molecular tag having adetectable property attached thereto or associated therewith a β and/orγ phosphate group and/or a base moiety of the NTP. In another preferredprobe, the NTP is a member selected from the group consisting of adeoxynucleotide triphosphate (dNTP), a nucleotide triphosphate (NTP) andanalogs thereof, particularly, where the NTP is a deoxynucleotidetriphosphate (dNTP), especially, where the deoxynucleotide triphosphate(dNTP) is a member selected from the group consisting of deoxyadenosinetriphosphate, deoxycyto sine triphosphate, deoxyguanosine triphosphateand deoxythymidine triphosphate. In another preferred probe, thenucleotide triphosphate (NTP) is a member selected from the groupconsisting of adenosine triphosphate, cytosine triphosphate, guanosinetriphosphate and uridine triphosphate. In another preferred probe, thetag is fluorophore, particularly, the fluorophore is a member selectedfrom the group consisting of fluorescein, 5carboxyfluorescein (FAM),rhodamine, 5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4,BODIPY dyes and cyanine dyes. In another preferred probe, the tag isattached to said β and/or γ-phosphate via a linker. In another preferredprobe, the fluorophore linker is an alkylene group having between about5 to about 12 carbons, particularly, where the fluorophore moiety is afluorescein or rhodamine dye.

The present invention relates to a kit for assaying pyrophosphatecleavage, said kit comprising: (a) a plurality of NTPs at least one NTPincluding an atomic and/or molecular tag or moiety havingdistinguishable/detectable property attached to and/or associated with aβ and/or γ-phosphate and/or a base moiety of the NTP and (b) apolymerase. In another preferred kit, the tag is a fluorophore and theproperty is fluorescence. In another preferred kit, the NTP furtherincludes a quencher attached to and/or associated with a β and/orγ-phosphate and/or a base moiety of the NTP, where each fluorophoreinteracts with said quencher moiety via a mechanism which is a memberselected from the group consisting of fluorescence resonance energytransfer (FRET), electron transfer and ground-state complex mechanism.

The present invention relates to a method for performing a primerextension reaction comprising the steps of (a) annealing anoligonucleotide primer to a portion of a template nucleic acid therebyforming a primer-template hybrid; (b) adding primer-extension reagentsincluding a tagged dNTP to afford increased or altered fidelity duringincorporation to the primer-template hybrid for extending the primer,where the tagged dNTP includes an atomic and/or molecular tag or moietyhaving distinguishable/detectable property attached to and/or associatedwith a β and/or γ-phosphate and/or a base moiety of the dNTP. The methodcan also include the step of adding cosubstrate-enzyme pair to theprimer-template hybrid for conducting a pyrophosphate-utilizing reactionin an amount sufficient to reduce peak dropout. In another preferredmethod, the cosubstrate-enzyme pair comprises pyrophosphate dependentphosphofructose kinase and fructose-6-phosphate. In another preferredmethod, the cosubstrate-enzyme pair comprises UDP GlucosePyrophosphorylase and UDP Glucose.

The present invention relates to a kit for performing a primer extensionreaction comprising: primer extension reagents and at least one dNTPincluding an atomic and/or molecular tag or moiety attached to and/orassociated with a β and/or γ-phosphate and/or a base moiety of the dNTPto increase or alter extension fidelity. The kit can further comprise acompound present in an amount sufficient to reduce peak dropout.

The present invention relates to a primer extension solution for theextension of a primer member of a primer template hybrid comprising:primer extension reagents at least one dNTP including an atomic and/ormolecular tag or moiety attached to and/or associated with a β and/orγ-phosphate and/or a base moiety of the dNTP to increase or alterextension fidelity. The solution can further comprise a compound presentin an amount sufficient to reduce peak dropout. In another preferredsolution, the cosubstrate-enzyme pair comprises pyrophosphate dependentphosphofructose kinase and fructose-6-phosphate. In another preferredsolution, the cosubstrate-enzyme pair comprises UDP GlucosePyrophosphorylase and UDP Glucose.

The present invention relates to a method of inhibiting or preventingpyrophosphorolysis during synthesis of a nucleic acid molecule, saidmethod comprising (a) combining one or more tagged nucleotides and anucleic acid template, where the tagged nucleotide comprises an atomicand/or molecular tag or moiety attached to and/or associated with a βand/or γ-phosphate and/or a base moiety of the nucleotide; and (b)incubating the one or more nucleotides and nucleic acid templatetogether with a polymerase and an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with alcoholgroup as acceptor, a nucleotidyltransferase, and a carboxy-lyase, underconditions sufficient to form a second nucleic acid moleculecomplementary to all or a portion of the nucleic acid template.

The present invention relates to a method of inhibiting or preventingpyrophosphorolysis during synthesis of a nucleic acid molecule, saidmethod comprising (a) combining a primer with a nucleic acid templateunder conditions sufficient to form a hybridized product; and (b)incubating said hybridized product in the presence of (i) one or moretagged nucleotides comprises an atomic and/or molecular tag or moietyattached to and/or associated with a β and/or γ-phosphate and/or a basemoiety of the nucleotide (ii) a polymerase, and (iii) an enzyme selectedfrom the group consisting of a pentosyltransferase, a phosphotransferasewith an alcohol group as acceptor, a nucleotidyltransferase, and acarboxy-lyase under conditions sufficient to synthesize a second nucleicacid molecule complementary to all or a portion of said nucleic acidtemplate.

The present invention relates to a method to prevent inhibition ofnucleic acid synthesis during amplification of a double stranded nucleicacid molecule, comprising (a) providing a first and second primer,wherein said first primer is complementary to a sequence at or near the3′ termini of the first strand of said nucleic acid molecule and saidsecond primer is complementary to a sequence at or near the 3′ terminiof the second strand of said nucleic acid molecule; (b) hybridizing saidfirst primer to said first strand and said second primer to said secondstrand in the presence of (i) a polymerase, and (ii) one or more taggednucleotides comprises an atomic and/or molecular tag or moiety attachedto and/or associated with a β and/or γ-phosphate and/or a base moiety ofthe nucleotide under conditions such that a third nucleic acid moleculecomplementary to said first strand and a fourth nucleic acid moleculecomplementary to said second strand are synthesized; (c) denaturing saidfirst and third strand and said second and fourth strand; and (d)repeating steps (a) to (c) one or more times.

The method of claim 47, wherein the hybridizing is in the presence of anenzyme selected from the group consisting of a pentosyltransferase, aphosphotransferase with an alcohol group as an acceptor, anucleotidyltransferase and a carboxy-lyase.

The present invention relates to a method of identifying a base at atarget position in a sample DNA sequence wherein an extension primer,which hybridises to the sample DNA either immediately adjacent to orvery near (within about 10 bases) to the target position is provided andthe sample DNA and extension primer are subjected to a polymerasereaction in the presence of a tagged deoxynucleotide ordideoxynucleotide, where the tagged deoxynucleotide or dideoxynucleotidean atomic and/or molecular tag or moiety having a detectable propertyattached to and/or associated with a β and/or γ-phosphate and/or a basemoiety of the deoxynucleotide or dideoxynucleotide, whereby the taggeddeoxynucleotide or dideoxynucleotide will only become incorporated andrelease pyrophosphate (PPi) if it is complementary to the base in thetarget position, any release of PPi being detected, differentdeoxynucleotides or dideoxynucleotides being added either to separatealiquots of sample-primer mixture or successively to the samesample-primer mixture and subjected to the polymerase reaction toindicate which deoxynucleotide or dideoxynucleotide is incorporated,characterised in that, a nucleotide-degrading enzyme is included duringthe polymerase reaction step, such that unincorporated nucleotides aredegraded.

In another preferred method, the nucleotide-degrading enzyme is apyrase.In another preferred method, the mixture of nucleotide-degrading enzymesis used having nucleoside triphosphatase, nucleoside diphosphatase andnucleoside monophosphatase activity. In another preferred method, thenucleotide-degrading enzyme is immobilised on a solid support. Inanother preferred method, the immobilised nucleotide-degrading enzyme isadded after nucleotide incorporation by the polymerase has taken place,and then removed prior to a subsequent nucleotide incorporation reactionstep. In another preferred method, the PPi release is directly detectedvia the detectable property of the tag. In another preferred method, thepolymerase reaction and PPi release detection steps are performedsubstantially simultaneously. In another preferred method, the sampleDNA is immobilised or provided with means for attachment to a solidsupport. In another preferred method, the sample DNA is first amplified.In another preferred method, the extension primer contains a loop andanneals back on itself and the 3′ end of the sample DNA. In anotherpreferred method, a native polymerase, an exonuclease deficient (exo-)high fidelity polymerase or a genetically modified polymerase is used.

In another preferred method, the method can be used for identificationof a base in a single target position in a DNA sequence wherein thesample DNA is subjected to amplification; the amplified DNA isimmobilized and then subjected to strand separation, the non-immobilizedstrand being removed and an extension primer, which hybridizes to theimmobilized DNA immediately adjacent to the target position, isprovided; each of four aliquots of the immobilized single stranded DNAis then subjected to a polymerase reaction in the presence of a taggeddeoxynucleotide, each aliquot using a different deoxynucleotide wherebyonly the tagged deoxynucleotide complementary to the base in the targetposition becomes incorporated.

The method can further comprising adding the identified dNTP to thethree non-extended chambers and repeating the cyclic identificationprocess.

The present invention relates to a kit for use in a method as defined inany one of claims 49 to 6.10, comprising: (a) a test specific primerwhich hybridizes to sample DNA so that the target position is directlyadjacent to the 3′ end of the primer; (b) a polymerase; and (c) at leastone tagged dNTP an atomic and/or molecular tag or moiety having adetectable property attached to and/or associated with a β and/orγ-phosphate and/or a base moiety of the dNTP. The kit can be used foruse with initial PCR amplification, further comprising: (i) a pair ofprimers for PCR, at least one primer having means permittingimmobilization of said primer; (ii) a polymerase for PCR; (iii) amixture of dNTPs including at least one tagged dNTP. The methods or kitscan also be used with a multiplicity of sample DNA sequences, whereinsaid DNA sequences are arranged in array format on a solid surface.

The present invention relates to a composition comprising adeoxyribonucleoside or ribonucleoside including a molecular and/oratomic tag attached to or associated with a β and/or γ phosphate group,a base moiety, and/or a sugar moiety, where the tag alters fidelity ofbase incorporation.

The present invention relates to a method comprising the step of addinga composition comprising a deoxyribonucleoside or ribonucleosideincluding a molecular and/or atomic tag attached to or associated with aβ phosphate group, a base moiety, and/or a sugar moiety, where the tagalters fidelity of base incorporation to a nucleotide polymerizationmedium comprising a nucleotide polymerase.

The present invention relates to a composition comprising a nucleotideor nucleotide analogs including a molecular and/or atomic tag on a βphosphate group and/or a base moiety adapted to increase the fidelity ofviral replication. In another preferred composition, the virus is HIV.

The present invention relates to a method for increasing the fidelity ofviral replication comprising administering an therapeutically effectiveamount of a nucleotide including a molecular and/or atomic tag on a βphosphate group and/or a base moiety to an animal including a human,where the nucleotide is designed to increase base incorporation fidelityduring viral replication. In another preferred method, the virus is HIV.

The present invention relates to a composition comprising a viralreplication fidelity enhancing agent selected from the group consistingof compounds of the following general formulas or mixtures orcombinations thereof:

Z—OPO₂O—Z′  (a)

Z—PO₂O—Z′  (b)

Z—OPO₂—Z′  (c)

Z—PO₂—Z′  (d)

Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)

Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)

Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)

Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h)

where Z or Z′ is a hydrogen atom or a thermally stable substituentcomprising primarily one or more atoms selected from the group carbon,nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atomsto satisfy valence requirements, E and E′ are an oxygen atom or athermally stable substituent comprising primarily one or more atomsselected from the group carbon, nitrogen, oxygen, sulfur and phosphoruswith sufficient hydrogen atoms to satisfy valence requirements and n isan integer having a value between 0 and about 5, and where the agent isadapted to increase the fidelity of viral replication. In anotherpreferred composition, the virus is HIV.

The present invention relates to a method for increasing the fidelity ofviral replication comprising administering to an animal including ahuman a therapeutically effective amount of a viral replication fidelityenhancing agent selected from the group consisting of compounds of thefollowing general formulas or mixtures or combinations thereof:

Z—OPO₂O—Z′  (a)

Z—PO₂O—Z′  (b)

Z—OPO₂—Z′  (c)

Z—PO₂—Z′  (d)

Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)

Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)

Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)

Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h)

where Z or Z′ is a hydrogen atom or a thermally stable substituentcomprising primarily one or more atoms selected from the group carbon,nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atomsto satisfy valence requirements, E and E′ are an oxygen atom or athermally stable substituent comprising primarily one or more atomsselected from the group carbon, nitrogen, oxygen, sulfur and phosphoruswith sufficient hydrogen atoms to satisfy valence requirements and n isan integer having a value between 0 and about 5, where the nucleotide isdesigned to increase base incorporation fidelity during viralreplication. In another preferred method, the virus is HIV.

The present invention also relates to biological memory storage andretrieval systems where the fidelity of the storage and retrieve processis improved by using fidelity enhances described herein. The methodwould include the step of synthesizing a sequence of monomerscorresponding to a given data sequence using the fidelity enhancingagent of this invention. Once the information is stored, the informationcan be retrieved by sequencing the sequence to retrieve the datasequence.

The present invention also relates to agents and methods forameliorating symptoms of animals including humans infected with aretrovirus, including the step of administering to the animal atherapeutically effective amount of a composition including a dNTPhaving an atomic and/or molecular tag, preferably, an atomic ormolecular tag on β and/or γ-tagged phosphate of the dNTP, to increasethe fidelity of the viruses reverse transcriptase, decrease mutation,increase the immune response to the virus, increase the effectiveness ofmedications to the virus and ameliorate symptoms associated with theviral infection.

The present invention also relates to agents and methods forameliorating symptoms of animals including humans suffering from cancer,including the step of administering to the animal a therapeuticallyeffective amount of a composition including a dNTP having an atomicand/or molecular tag, preferably, an atomic or molecular tag on β and/orγ-tagged phosphate of the dNTP, to increase the fidelity of thepatient's natural polymerases, decrease mutations, increase the immuneresponse to the cancer, increase the effectiveness of medications to thecancer and ameliorate symptoms associated with the cancer.

The present invention also relates to agents and methods forameliorating symptoms of aging in animals including humans, includingthe step of administering to the animal a therapeutically effectiveamount of a composition including a dNTP having an atomic and/ormolecular tag, preferably, an atomic or molecular tag on β and/orγ-tagged phosphate of the dNTP, to increase the fidelity of thepatient's natural polymerases, decrease mutations, increase cellularvitality, and ameliorate symptoms of aging.

The present invention also relates to agents and methods for reducingthe evolutionary tendencies of retro virus such as HIV. HIV-1, thecausative agent of AIDS, has evolved many ways to defeat its human hostdefenses. One of these ways involves evading the immune system byinaccurately replicating its genome (one mistake per 2,000-5,000 bases).The polymerase responsible for the inaccurate replication is HIV-1reverse transcriptase (RT). RT converts the single stranded RNA genomeinto a complementary DNA strand, destroys the RNA template, and uses thenascent DNA strand to template synthesis of the double-stranded DNAversion of the HIV-1 genome. Since the HIV genome is approximately10,000 bases, this error-prone process produces a variant genomeessential every time the virus replicates. The misincorporated bases canspecify altered HIV protein sequences. Thus, the immune system in apatient infected with HIV is fighting a losing battle, since viralproteins (antigens) are constantly changing. Additionally, theactivities of these protein variants may be modified and, if the patientis following a drug-treatment therapy, drug-resistant variants mayemerge due to selective pressures. Thus, virus evolution mediatedthrough inaccurate genome replication is a significant problem, bothwith HIV-1 and with any virus whose replication is mediated by anerror-prone polymerase.

The fidelity of HIV-1 RT is improved in vitro by providing the enzymewith nucleotides containing a molecular tag on the γ-phosphate. Thisunexpected discovery may lead to a novel therapeutic that willneutralize the genetic mutability of this deadly virus. Understandingthe mechanism by which RT selects nucleotides for incorporation willproduce insights into enzymatic DNA synthesis and evolution of viraldiversity. Ultimately, a novel therapeutic that increases enzymefidelity may minimize antigen evolution, enabling the immune system toeliminate virus and virus infected cells, and minimize the emergence ofdrug resistance. Understanding why improved accuracy is observed in thecontext of the modified nucleotide may enable design a small moleculethat has this same effect, but that would be more easily delivered intocells.

Fidelity

The inventors have found that novel nucleotides can be prepared thatimprove fidelity of incorporations where the nucleotides include acovalently attached substituent on β and/or γ phosphate of a NTP, dNTPor ddNTP where the substituent includes a aminonaphthalene-1-sulfonate(ANS) group. The tagged nucleotide and preferably the tagged γ-phosphateof the nucleotide improves the fidelity at which this nucleotide analogis incorporated by commercially available Taq DNA polymerase.

Pyrophosphorolysis Inhibition

Addition of pyrophosphatase to a polymerase chain reaction greatlyenhances the progress of that reaction, and provides superior resultscompared to use of the method without a pyrophosphatase (Tabor andRichardson, 1996). Similarly addition of a pyrophosphatase to a DNAsequencing reaction provides more uniformity in intensities of bandsformed in a polyacrylamide gel used to identify products of thesequencing reaction pyrophosphatase (Tabor and Richardson, 1996). Thisuniformity is thought to be due to prevention of degradation of specificDNA products via pyrophosphorolysis. Any modification to the nucleotidethat is capable of inhibiting the pyrophosphorolysis reaction is usefulin this invention. One way to inhibit pyrophosphorolysis is to breakdown any pyrophosphate that is generated during a polymerase reaction,by adding the enzyme pyrophosphatase. Even trace addition of apyrophosphatase (one thousandth the molar ratio of DNA polymerasemolecules in a solution) to a primer extension reaction completelystabilizes oligonucleotide fragments produced in a polymerase reaction,by preventing pyrophosphorolysis. The agent should be added at aconcentration sufficient to either catalyze the hydrolysis ofpyrophosphate in the reaction mixture at a rate that will preventaccumulation of pyrophosphate to a level that will lead topyrophosphorolysis, or prevent accumulation of pyrophosphate in anyother manner. The amount of agent needed is readily determined bystandard techniques. However, the inventors have discovered thatpyrophosphorolysis can also be reduced or eliminated by usingnucleotides containing molecular and/or atomic substituents on the βand/or γ phosphate moieties.

Nucleic Acid Sequencing Using Tagged PP_(i) Detection

In certain embodiments, the present invention provides a heterogeneousassay for the detection of released tagged pyrophosphate. The detectionof tagged pyrophosphate is advantageous in a number of biologicalreactions. For example, in a DNA polymerase reaction, single molecule orbulk, wherein the polymerase selects a single DNA molecule from solutionand thereafter incorporates the nucleotide at the 3′-end of a primerstrand, the natural consequence of such incorporation is the release ofpyrophosphate. If the assay solution comprises the four deoxynucleotidetriphosphates, each dNTP labeled with a different molecular and/oratomic tag such as a fluorescent dye having a different color attachedto the β- and/or γ-phosphate, it is then possible to sequentially recordthe activity of the polymerase operating on a target DNA. The nucleotidesequence of the target DNA can thereafter be directly read from theorder of released dyes attached to the pyrophosphate. If the assaysolution comprises the four deoxynucleotide triphosphates, each dNTPlabeled with a different molecular and/or atomic tag such as afluorescent dye having a different color attached to the β- and/orγ-phosphate and activating tags bonded to or associated with thepolymerase or other species in the medium, it is then possible also tosequentially record the activity of the polymerase operating on a targetDNA. The nucleotide sequence of the target DNA can thereafter be readdirectly from the order of released dyes attached to the pyrophosphate.

As such, the present invention provides a heterogeneous assay method fordetecting pyrophosphate release, the components of the assay comprisinga labeled NTP, a target nucleic acid, a primer nucleic acid and apolymerase, the method comprising: (a) flowing the labeled nucleotidetriphosphate (NTP) having a molecular and/or atomic tag bonded to orassociated with a β- and/or γ-phosphate moiety of the NTP, past animmobilized component selected from the group consisting of thepolymerase, the primer and the target nucleic acid; (b) incorporatingthe tagged dNTP on a primer strand hybridized to the target nucleic acidusing an enzyme and releasing the γ-phosphate with the fluorophoremoiety attached thereto; and (c) detecting the fluorescent moietythereby detecting NTP binding, incorporation and/or pyrophosphatecleavage. In the heterogeneous assay of the present invention, eitherthe polymerase, the primer or the target nucleic acid is attached to asolid phase, such as a solid support. Preferably, in the methods of thepresent invention, the polymerase is immobilized on a solid support.

In certain aspects, the polymerase is a DNA polymerase such as DNApolymerase I, II or III. In other aspects, suitable polymerases include,but are not limited to, a DNA dependent RNA polymerase and reversetranscriptase such as an HIV reverse transcriptase. Specific examplesinclude, but are not limited to, T7 DNA polymerase, T5 DNA polymerase,E. coli DNA polymerase I, T4 DNA polymerase, T7 RNA polymerase and TaqDNA polymerase. Those of skill in the art will know of other enzymes orpolymerases suitable for use in the present invention. In certainaspects, the polymerase is bathed in a flowing solution comprising:unlabeled, single-stranded DNA fragments hybridized to anoligonucleotide primer and a mixture of NTPs.

In certain aspects of the present invention, a labeled nucleotidetriphosphate (NTP) having a molecular and/or atomic tag bonded to orassociated with a β- and/or γ-phosphate moiety of the NTP isincorporated into a polynucleotide chain. The dNTP incorporation into agrowing oligonucleotide by a DNA polymerase results in pyrophosphaterelease. In this reaction, the phosphate ester bond between the α and βphosphates of the incorporated nucleotide is cleaved by the DNApolymerase, and the β- and/or γ-phosphate moieties of the resultingpyrophosphate are released in solution. As used herein, the termpyrophosphate also includes substitution of any of the oxygen atoms ofthe pyrophosphate group with an atom that enables attachment of themolecular moiety that will be detected and provide information about theidentity of the incorporated nucleotide, a nitrogen or a sulfur atom orcombinations thereof to generate azapyrophosphate, diazapyrophosphte,thiopyrophosphate, dithiopyrophosphate, etc.

If the tag is a fluorophore, then the fluorophore can be detected eitherupon nucleotide binding, during incorporation or after the nucleotideand the pyrophosphate are released. In certain aspects, release of thepyrophosphate caused by cleavage of the α-β bond can switch thefluorophore moiety into a fluorescent state either by fluorophoredequenching or fluorophore activation. This event can then be detectedusing an ultrasensitive fluorescence detector. Using single moleculedetection for example, fluorescent signals appear at the locations ofthe individual molecules being observed. In certain aspects, each typeof nucleotide is labeled with a different fluorophore so that theincorporated nucleobases can be sequentially identified by thefluorophores during binding, incorporation or release. Preferably, thedeoxy nucleotide triphosphates (dNTPs) of the present methods include,but are not limited to, deoxyadenosine triphosphate, deoxycytosinetriphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate,deoxyuridine triphosphate or mixtures thereof, each with a uniquemolecular and/or atomic tag attached to the β- and/or γ-phosphate moietyof the NTP.

As is described in detail hereinbelow, the nucleotides of the presentinvention, both purine and pyrimidine varieties, are modified at varioussites with a molecular and/or atomic tag such as a fluorophore orchromophore. In certain aspects, the fluorophore or chromopore aredesigned to interact with other tags situated on specific sites of thepolymerase or associated with other agents in the medium. Once thetagged dNTPs are produced, they can be used to sequence DNA strands bydirect single molecule detection. The tags can be detected when thelabeled dNTP binds to the polymerase, during incorporation or uponrelease by measuring a detectable property of the tag alone or as aresult of an interaction with another tag associated with other agent inthe medium including the polymerase itself. The detectable property canof course be fluorescence or induced fluorescence. The ultrasensitivityof the present methods provide unprecedented economy and representsubstantial improvements over the methods of the prior art.

The tagged dNTPs and formed tagged pyrophosphates can be used in singlemolecule detection formats. In certain embodiments, an unlabeled,single-stranded target nucleic acid with a primer hybridized thereto istethered to the surface of a solid support such as a glass slide. Anaqueous solution comprising an enzyme, such as a DNA polymerase, andtagged dNTPs flows across the surface. In another embodiment, anindividual polymerase molecule is immobilized on a glass slide and thepolymerase is bathed in a flowing solution comprising: 1) unlabeled,single-stranded DNA fragments hybridized to an oligonucleotide primerand 2) a mixture of tagged deoxynucleotide triphosphates. In yet anotherembodiment, a library of oligonucleotides can be immobilized on a solidsupport such as glass and the glass is bathed in a solutioncomprising: 1) a polymerizing agent such as a polymerase, 2) unlabeled,single-stranded DNA fragments hybridized to an oligonucleotide primerand 3) a mixture of tagged deoxynucleotide triphosphates. In a furtherembodiment, an individual polymerase molecule is immobilized on a glassslide and the polymerase is bathed in a solution comprising: 1) nickeddouble strained DNA, where the nicking is either affected via chemicalmeans such as Fe-EDTA or via enzymatic means such as DNase, and 2) amixture of tagged deoxynucleotide triphosphates.

If the tags are capable of fluoresceing or luminesceing, then anevanescent light field is set up by total internal refection (TIR) of alaser beam at the glass-aqueous solution interface. In certain aspects,the TIR illumination field is continuously imaged at video-rate with aCCD camera or an intensified charge couple device (ICCD) camera.

Solid Phase

The present invention relates to a heterogenous assay wherein a materialin the solid-phase interacts with reagents in the liquid phase. Incertain aspects, the nucleic acid is attached to the solid phase. Thenucleic acid can be in the solid phase such as immobilized on a solidsupport, through any one of a variety of well-known covalent linkages ornon-covalent interactions. The support is comprised of insolublematerials, such as controlled pore glass, a glass plate or slide,polystyrene, acrylamide gel and activated dextran. In other aspects, thesupport has a rigid or semi-rigid character, and can be any shape, e.g.,spherical, as in beads, rectangular, irregular particles, gels,microspheres, or substantially flat, so long as the support permitssingle molecule detection. In some embodiments, it can be desirable tocreate an array of physically separate sequencing regions on the supportwith, for example, wells, microtubes or nanotubes derivatived to capturepart of the DNA sequencing complex/enzyme, primer or template such ashistidine 5′ derivation, or other random modification so that thecomplex can stick to the tubes, raised regions, dimples, trenches, rods,pins, inner or outer walls of cylinders, and the like. Other suitablesupport materials include, but are not limited to, agarose,polyacrylamide, polystyrene, polyacrylate, hydroxethylmethacrylate,polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts ofsuch. Other embodiments of solid-supports include small particles,non-porous surfaces, addressable arrays, vectors, plasmids, orpolynucleotide-immobilizing media.

As used in the methods of the present invention, nucleic acid can beattached to the solid support by covalent bonds, or other affinityinteractions, to chemically reactive functionality on thesolid-supports. The nucleic acid can be attached to solid-supports attheir 3′, 5′, sugar, or nucleobase sites. In certain embodiments, the 3′site for attachment via a linker to the support is preferred due to themany options available for stable or selectively cleavable linkers.Immobilization is preferably accomplished by a covalent linkage betweenthe support and the nucleic acid. The linkage unit, or linker, isdesigned to be stable and facilitate accessibility of the immobilizednucleic acid to its sequence complement. Alternatively, non-covalentlinkages such as between biotin and avidin or stepavidin are useful.Examples of other functional group linkers include ester, amide,carbamate, urea, sulfonate, ether, and thioester. A 5′ or 3′biotinylated nucleotide can be immobilized on avidin or strepavidinbound to a support such as glass.

In other aspects of the heterogenous assay of the present invention, thepolymerase is immobilized on a solid support. Suitable solid supportsinclude, but are not limited to, controlled pore glass, a glass plate orslide, polystyrene, and activated dextran. In other aspects, syntheticorganic polymers such as polyacrylamide, polymethacrylate, andpolystyrene are also illustrative support surfaces. In addition,polysaccharides such as cellulose and dextran, are further illustrativeexamples of support surfaces. Other support surfaces such as fibers arealso operable.

In other aspects, polymerase immobilization is accomplished using solidchromatography resins, that have been modified or activated to includefunctional groups that permit the covalent coupling of resin to enzyme.Typically, aliphatic linker arms are employed. The enzymes of thepresent invention can also be noncovalently attached to a solid supportsurface through, for example, ionic or hydrophobic mechanisms.

Covalent attachment of a protein or nucleic acid to a glass or metaloxide surface can be accomplished by first activating the surface withan amino silane. DNA or protein derivatized with amine-reactivefunctional groups can then attach to the surface (see, K. Narasimhan etal., Enzyme Microb. Technol. 7, 283 (1985); M. J. Heller et al., U.S.Pat. No. 5,605,662; and A. N. Asanov et al., Anal. Chem. 70, 1156(1998)).

The ordinarily skilled artisan will know numerous other schemes forlinking nucleic acid and proteins to support surfaces. Moreover, thechoice of support surface and the method of immobilizing the enzyme islargely a matter of convenience and depends on the practitioner'sfamiliarity with, and preference for, various supports surfaces, as wellas preference for various immobilizing schemes, and knowledge of thesubstrate.

In assay operation, the enzyme, such as a DNA polymerase, selects asingle DNA molecule from solution. The polymerase incorporates a firstnucleotide at the 3′-end of the primer strand and releases therespective PP₁. The polymerase then translocates to the next position onthe target DNA, incorporates a complementary tagged nucleotide, andreleases the respective pyrophophate. The tagged nucleotide can bedetected upon binding to tagged polymerase, upon incorporation by taggedpolymerase, and/or upon release of the tagged pyrophosphate eitherdirectly or as a result of interaction with another tag on an agent inthe medium. These events can then be recorded sequentially using adetection system capable of detecting a detectable property of the tagsuch as by video-rate imaging using for example, a CCD or ICCD camera,capable of detecting fluorescence from a single tag where the tag isfluorophore or a chromophore. The resulting movie shows the activity ofa single polymerase molecule operating on a single molecule of DNA. Thenucleotide sequence of the DNA target is read directly from the order ofbase incorporation by detecting the tag during base binding, baseincorporation and/or pyrophosphate release. Each of those events orsteps during incorporation provides information about the process and aunique pattern is associated with each nucleotide. The match of eachbase incorporation pattern is used to increase confidence of each basecall. Time, intensity and wavelength or frequency are each monitored toprovide maximal confirmatory information.

When the first nucleic acid molecule has been sequenced, the polymerasereleases it and selects another template from solution. Many DNAmolecules are thereby sequenced by a single polymerase. The processcontinues for the life of the enzyme or more specifically, the life ofthe interacting tag within the enzyme.

To minimize signals that are not optimally positioned, assays usingimmobilized polymerase are preferred because once the detector systemsuch as a CCD or ICCD camera is focused on the plane containing thepolymerase, the focus will not have to be changed during a sequencingrun. Otherwise, the plane of focus may need to be changed due totranslocation of the polymerase through the medium as polymerizationproceeded. Not only is changing the focal plane more difficult, thetranslocation of the polymerizing sites could result in a change in thenumber of polymerizing sites within a given viewing field over time andadversely affect data integrity. Moreover, the lengths of the DNAtemplates should preferably be significantly uniform (±10%),substantially uniform (±5%) or essentially uniform (±1%) in length tofurther maximize signal detection from the replication complexes.

Since there are approximately 3.4 angstroms between base pairs, a 1000bases synthesis would involve approximately 3400 angstroms movement ofeither DNA through the polymerase or polymerase along the DNA. Thus, ifthe polymerase is immobilized and DNA is passed through the polymerase,signal remains localized at the position of the polymerase. If, however,the template or primer is immobilized, the signal produced duringincorporation may move by the distance of the sequence read length.Therefore, if the DNA primer or template is immobilized, in order tominimize or eliminate overlap between the sequencing complexes it ispreferred that the immobilized molecules are separated by a distance of10 times, 5 times, or approximately the distance of the desired sequenceread. By so doing, the essentially random motion of the extendingstrands that results from their presence in the polymerizing solutionwill not interfere with neighboring sequencing complexes.

Of course, computer programs could be written to analyze the data overtime and correct for most of the adverse affects of polymerasetranslocation. Moreover, for sparsely populated sequencing reactions,the likelihood of changes in the number of active polymerizing sites perview field can be reduced.

Preparation of Target Nucleic Acid

The target nucleic acid can be prepared by various conventional methods.For example, target nucleic acid can be prepared as inserts of any ofthe conventional cloning vectors, including those used in conventionalDNA sequencing. Extensive guidance for selecting and using appropriatecloning vectors is found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989), and like references. Sambrook et al. and Innis et al,editors, PCR Protocols (Academic Press, New York, 1990) also provideguidance for using polymerase chain reactions to prepare targetpolynucleotides. Cloned or PCR amplified target nucleic acid is preparedwhich permit attachment to solid supports.

Preferably, the target nucleic acid sequences are from sheared DNAfragments from a subject organism, preferably human, and are treated toprovide blunt ends, then ligated to two oligodeoxynucleotides. Forexample, one oligonucleotide can be derivatized with biotin at its 5′ or3′ end but the 5′ end is preferred since that will cause fewer stericproblems. Further, the first primer may be 5′ biotinylated and thesecond is complementary to the biotinylated primer and contains a 5′phosphate. The ligated DNA is denatured, it is brought into contact witha streptavidin-activated slide, and it attaches through the 5′ biotin tothe slide. A primer is hybridized to the tethered fragments prior tosequencing. This sequencing primer is the same sequence as thebiotinylated primer. Only DNA fragments having each type of ODN can bothattach and be sequenced; fragments having two phosphorylated primerswill not attach.

DNA attachment could also be accomplished by direct covalent coupling aspracticed on DNA chips (see, U.S. Pat. No. 5,605,662). Unlike DNA chipsthat require a dense lawn of probes, preferably, a few DNA molecules arebound per unit surface area. Binding density is easily controlled byadding a carrier to the DNA sample (e. g., free biotin to a biotinylatedDNA sample).

Detection

The tagged NTP can be detected by a variety of analytical techniques. Ifthe tags are atomic or molecular tags with characteristic NMR, MS and/orother physical or chemical tag response signals, then the reaction canbe monitored in real time using pulsed NMR techniques, MS techniques ortechniques associated with other physical and/or chemical tag responses.The tags can even be shift reagents. If the tags are molecules thatinteract with other molecules in the presence of light to produce afluorescent signature, then the reaction can be monitored usingfluorescent spectroscopy on a continuous or discrete format. It shouldbe recognized that tags can be prepared that have any desired detectableproperty.

In certain embodiments, the enzymatic reaction is monitored using singlemolecule detection. The single-molecule fluorescence detection of thepresent invention can be practiced using optical setups includingnear-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, and total internal reflection fluorescence(TIRF) microscopy. Suitable photon detectors include, but are notlimited to, photodiodes and intensified CCD cameras. In a preferredembodiment, an intensified charge couple device (ICCD) camera is used.The use of a ICCD camera to image individual fluorescent dye moleculesin a fluid near the surface of the glass slide is advantageous forseveral reasons. With an ICCD optical setup, it is possible to acquire asequence of images (movies) of fluorophores. In certain aspects, each ofthe NTPs of the present invention has a unique fluorophore associatedwith it, as such, a four-color instrument can be used having fourcameras and four excitation lasers or beam-splitters may be used tomonitor fluorescent intensity changes at a number of desire frequencies.Thus, it is possible to use this optical setup to sequence DNA. Inaddition, many different DNA molecules spread on a microscope slide canbe imaged and sequenced simultaneously. Moreover, with the use of imageanalysis algorithms, it is possible to track the path of single dyes anddistinguish them from fixed background fluorescence and from“accidentally dequenched” dyes moving into the field of view from anorigin upstream.

In certain other embodiments, the sequencing works by directly detectingthe release tagged pyrophosphate, where a single dNTP is feed each timeand the polymerase is washed between before the next incorporation.

In certain aspects, the preferred geometry for ICCD detection of singlemolecules is total internal reflectance fluorescence (TIRF) microscopy.In TIRF, a laser beam totally reflects at a glass-water interface. Thefield does not end abruptly at the reflective interface, but itsintensity falls off exponentially with distance. The thin “evanescent”optical field at the interface provides low background and enables thedetection of single molecules with signal-to-noise ratios of 12:1 atvisible wavelengths (see, M. Tokunaga et al., Biochem. and Biophys. Res.Comm. 235, 47 (1997) and P. Ambrose, Cytometry, 36, 244 (1999)).

The penetration of the field beyond the glass depends on the wavelengthand the laser beam angle of incidence. Deeper penetrance is obtained forlonger wavelengths and for smaller angles to the surface normal withinthe limit of a critical angle. In typical assays, fluorophores aredetected within about 200 nm from the surface which corresponds to thecontour length of about 600 base pairs of DNA. Preferably, a prism-typeTIRF geometry for single-molecule imaging as described by Xu and Yeungis used (see, X-H. N. Xu et al., Science, 281, 1650 (1998)).

DNA, proteins and lipids have all been detected in complex samples withsingle-molecule sensitivity using labeled probes (see, L. Edman et al.,Proc. Natl. Acad. Sci. USA, 93, 6710 (1996); M. Kinjo et al., NucleicAcids Res. 23, 1795 (1995); A. Castro and J. G. K. Williams, Anal. Chem.69, 3915 (1997); S. Nie, et al., Science 266, 1018 (1994); S. Nie, etal., Anal. Chem. 67, 2849 (1995); and T. Schmidt et al., Proc. Natl.Acad. Sci. USA 9, 2926 (1996)). In addition to simple detection, singlefluorophores are also characterized with respect to fluorescencelifetime, spectral shifts and rotational orientation. In a preferredaspect of the present invention, an aqueous solution comprising anenzyme, such as a DNA polymerase, and distinguishable fluorogenic dNTPs,i.e., a characteristic dye for each nucleobase, flows across thesurface. An evanescent light field is set up by total internal refection(TIR) of a laser beam at the glass-aqueous solution interface. Incertain aspects, the TIR illumination field is continuously imaged atvideo-rate with an intensified charge couple device (ICCD) camera. It isthus possible to image the pyrophosphate as it is hydrolyzed by theenzyme.

Upon incorporation by polymerase, the tagged dNTP is hydrolyzed as usualand the liberated tagged pyrophosphate diffuses into the surroundingmedium. The tagged dNTP can be detected upon binding, incorporation orrelease or the free tagged pyrophosphate can be detected by detectingthe detectable property of the tag such as fluorescent and itsappearance is imaged at video-rate under a microscope. A flowing streamsweeps the dye away from the parent DNA molecule. As the DNA moleculecontinues to move through the polymerase due to the immobilizedpolymerase, the nucleotide sequence is read from the order of releaseddyes. Sequencing proceeds quickly, as fast as the polymerase progressesalong the DNA template.

In another embodiment, the present invention includes sensors asdisclosed in U.S. Pat. No. 5,814,524 which issued to Walt et al., onSep. 29, 1998, incorporated herein by reference. An optical detectionand identification system is disclosed therein that includes an opticsensor, an optic sensing apparatus and methodology for detecting andevaluating one or more analytes or ligands of interest, either alone orin mixtures. The system is comprised of a supporting member and an arrayformed of heterogeneous, semi-selective polymer films which function assensing receptor units and are able to detect a variety of differentanalytes and ligands using spectral recognition patterns. Using thissystem, it is possible to combine viewing and chemical sensing withimaging fiber chemical sensors.

High Throughput Screening

The present invention also provides integrated systems forhigh-throughput screening of DNA sequencing and pyrophosphate detection.The systems typically include robotic armature which transfers fluidfrom a source to a destination, a controller which controls the roboticarmature, an ICCD camera, a data storage unit which records thedetection, and an assay component such as a microtiter dish or asubstrate comprising a fixed reactant. A number of robotic fluidtransfer systems are available, or can easily be made from existingcomponents. For example, a Zymate XP (Zymark Corporation; Hopkinton,Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.)pipetting station can be used to transfer parallel samples to 96, 384 ormore welled microtiter plates to set up several parallel simultaneouspolymerase reactions.

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e. g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image. In certain aspects, the integrated system ofthe present invention carries light from the specimen field to thecharge-coupled device (CCD) camera, which includes an array of pictureelements (pixels). The light from the specimen is imaged on the CCDcamera. Particular pixels corresponding to regions of the specimen (e.g., individual polymerase sites on a glass surface) are sampled toobtain light intensity readings for each position. Multiple pixels areprocessed in parallel to increase speed. The apparatus and methods ofthe invention are easily used for viewing any sample, e.g., byfluorescent or dark field microscopic techniques.

There is a great deal of practical guidance available in the literaturefor providing an exhaustive list of fluorescent and chromogenicmolecules and their relevant optical properties (see, for example,Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2ndEdition (Academic Press, New York, 1971); Griffiths, Colour andConstitution of Organic Molecules (Academic Press, New York, 1976);Bishop, Ed., Indicators (Pergamon Press, Oxford, 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene, 1992) Pringsheim, Fluorescence and Phosphorescence (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing fluorophore and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleotide, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760, incorporated herein by reference.

Suitable donors and acceptors operating on the principle of fluorescenceenergy transfer (FRET) include, but are not limited to,4-acetamido-4′isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;-(4-anilino-1naphthyl) maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′diisothiocyanatostilbene-2,2′-disulfonic acid; 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DAB ITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline;

Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots;Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.

In certain embodiments, certain visible and near IR dyes are known to besufficiently fluorescent and photostable to be detected as singlemolecules. In this aspect the visible dye, BODIPY R6G (525/545), and alarger dye, LI-COR's near-infrared dye, IRD-38 (780/810) can be detectedwith single-molecule sensitivity and can be used to practice the presentinvention.

There are many linking moieties and methodologies for attachingfluorophore or quencher moieties to nucleotides, as exemplified by thefollowing references: Eckstein, editor, Oligonucleotides and Analogues:A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al.,NucleicAcidsResearch, 15: 5305-5321 (1987) (3′thiol group onoligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′phosphoamino groupvia Aminolink™ II available from Applied Biosystems, Foster City,Calif.) Stabinsky, U.S. Pat. No. 4,739,044 (3′aminoalkylphosphorylgroup); AP3 Labeling Technology (U.S. Pat. Nos. 5,047,519 and 5,151,507,assigned to E. I. DuPont de Nemours & Co); Agrawal et al., TetrahedronLetters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5′mercaptogroup); Nelson et al., NucleicAcidsResearch, 17: 7187-7194 (1989)(3′amino group); and the like.

Primer Extension Reaction

Generally, the primer extension reaction of the present inventioncomprises the following steps: (i) providing a template nucleic acid;(ii) annealing an oligonucleotide primer to a portion of the templatenucleic acid thereby forming a primer-template hybrid; (iii) addingprimer-extension reagents to the primer-template hybrid for extendingthe primer; and (iv) optionally adding a cosubstrate-enzyme pair to theprimer-template hybrid for conducting a pyrophosphate-utilizingreaction.

Any source of nucleic acid can be used as a template nucleic acidprovided it can be presented in a single stranded form and is capable ofannealing with a primer oligonucleotide. Exemplary template nucleicacids include DNA, RNA, which DNA or RNA may be single stranded ordouble stranded. More particularly, template nucleic acid may be genomicDNA, messenger RNA, cDNA, DNA amplification products from a PCRreaction, and the like. Methods for preparation of template DNA may befound elsewhere (ABI PRISM Dye Primer Cycle Sequencing Core Kit).Standard protocols for primer-template annealing and primer extension inthe context of PCR or Sanger-type sequencing may be found elsewhere(Innis; Deiffenbach; ABI PRISM Dye Primer Protocol; ABI PRISM DyeTerminator Protocol). Generally, to perform a primer extension reactionin the context of PCR, template nucleic acid is mixed with a pair of PCRprimers and primer-extension reagents comprising a buffer, MgCl₂,deoxynucleotide triphosphates or preferably for increase accuracy of theamplification products, β and/or γ tagged dNPTs, and a DNA polymerase.For example, a typical PCR reaction includes 20 pmol of each primer, 20mM buffer at pH 8, 1.5 mM MgCl₂, 10 to 500 preferably 200 of eachdeoxynucleotide triphosphate (dNTP), and 2 units of Taq polymerase orother suitable thermostable polymerase.

The reaction mixture is then thermocycled, a typical thermocycle profilecomprising a denaturation step (e.g. 96° C., 15 s), a primer annealingstep (e.g., 55° C., 30 s), and a primer extension step (e.g., 72° C., 90s). Typically, the thermocycle is repeated from about 10 to about 100cycles or more.

Kits and Solutions of the Invention

In another aspect, the present invention includes kits and solutions forperforming the primer extension methods of the invention. The kits andsolutions of the invention include primer extension reagents with themodified (tagged) dNTPs and/or modified PP_(i) and optionally acosubstrate-enzyme pair. Optionally, the kits may also include primers.The elements of the kits may be packaged in a single container ormultiple containers. In one preferred configuration, a polymerase enzymeand modified (tagged) dNTPs and/or modified PP_(i) are packaged in thesame container.

This invention may also be used in methods where improvement of theaccuracy of the synthesis of nucleic acids by a polymerase is desiredand where pyrophosphorolysis is deemed counter-productive. Uses include:polymerase chain reaction, especially ‘Long PCR,’ and cDNA synthesis.Examples of patents describing these methods include U.S. Pat. No.4,965,188, U.S. Pat. No. 5,079,352, U.S. Pat. No. 5,091,310, U.S. Pat.No. 5,142,033, U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, U.S.Pat. No. 4,800,159, U.S. Pat. No. 5,512,462 and U.S. Pat. No. 5,405,776,incorporated by reference. In the case of cDNA synthesis, a reversetranscriptase polymerase is incubated with the mRNA template and thetagged deoxynucleoside triphosphates.

The invention also relates to a kit comprising a container means, suchas a box, having in close confinement therein two or more containerssuch as vials, ampules, tubes, jars or the like, each of which containsthe materials necessary to carry out the invention. For example, acontainer may contain a polymerizing agent such as a DNA or RNApolymerase. Another container may contain a tagged phosphate,pyrophosphate or polyphosphate in an amount sufficient to reduce,inhibit or prevent pyrophosphorolysis. And other containers may containtagged and/or untagged dNTPs. Alternatively, the kit can include acontainer containing a polymerizing agent such as a DNA or RNApolymerase and other containers containing tagged and/or untagged dNTPs.

Preferably, the contents of the containers are present at workingconcentrations or requiring a to fold dilution to achieve workingconcentrations. Other containers may contain other reagents that arenecessary for carrying out dideoxy sequencing or amplification (PCR).

Thus, the solution of the present invention is an aqueous and/orbuffered liquid containing the components described above. Thesecomponents are present in the solution at concentrations sufficient toperform their desired function. If the reaction mixture is intended toamplify a target nucleic acid molecule, the reaction mixture willcontain the tagged dNTPs which reduce the level of pyrophosphate andincrease polymerase fidelity, a polymerizing agent such as a DNApolymerase, all four dNTPs, and one or more oligonucleotide primershaving a single stranded region which are capable of annealing to thetarget nucleic acid molecule, being extended and thereby amplified. Theprimer extension reaction may also comprise a chain terminator asdescribed herein, e.g., a dideoxynucleoside triphosphate and the ddNTPmay be modified at the β and/or γ phosphate to reduce background and/orreduce band spreading, which allows for sequencing of the target DNAmolecule by the well known Sanger dideoxy sequencing method.

The present invention uses tagged NTPs that having molecular and/oratomic tags bonded to or associated with the β and γ phosphates so asnot to alter the chemistry of the growing polymer chain, allow fordetection of the NTPs (including dNTPs and ddNTPs) upon binding, duringincorporation and after incorporation via the released taggedpyrophosphate. Optionally, the present invention can includephosphatases that degrade NTPs (including dNTPs and ddNTPs) or thatdegrade pyrophosphate. However, when the labeled pyrophosphates act toinhibit pyrophosphorolysis, then such degradation enzymes should betailored to attach the NTPs and not the tagged pyrophosphate.

Detailed nucleoside di- and/or mono-phosphate degrading enzymes areoptional and may be used in combination with a nucleoside tri-phosphatedegrading enzyme. Suitable such enzymes include most notably apyrasewhich is both a nucleoside diphosphatase and triphosphatase, catalyzingthe reactions NTP-NMP+2Pi and NTP-NDP+Pi (where NTP is a nucleosidetriphosphate, NDP is a nucleoside diphospate, NMP is a nucleotidemonophosphate and Pi is phosphate). Apyrase may be obtained from SigmaChemical Company. Other suitable nucleotide triphosphate degradingenzymes include Pig Pancreas nucleoside triphosphate diphosphohydrolase(Le Bel et al., 1980, J. Biol. Chem., 255, 1227-1233). Further enzymesare described in the literature.

Different combinations of nucleoside tri-, di- or monophosphatases maybe used. Such enzymes are described in the literature and differentenzymes may have different characteristics for deoxynucleotidedegradation, e.g., different Km, different efficiencies for a differentnucleotides etc. Thus, different combinations of nucleotide degradingenzymes may be used, to increase the efficiency of the nucleotidedegradation step in any given system. For example, in some cases, theremay be a problem with contamination with kinases which may convert anynucleoside diphosphates remaining to nucleoside triphosphates, when afurther nucleoside triphosphate is added. In such a case, it may beadvantageous to include a nucleoside disphosphatase to degrade thenucleoside diphosphates. Advantageously all nucleotides may be degradedto nucleosides by the combined action of nucleoside tri-, di- andmonophosphatases.

Generally speaking, the nucleotide-degrading enzyme is selected to havekinetic characteristics relative to the polymerase such that nucleotidesare first efficiently incorporated by the polymerase, and then anynon-incorporated nucleotides are degraded. Thus, for example, ifdesired, the K_(m) of the nucleotide-degrading enzyme may be higher thanthat of the polymerase such that nucleotides which are not incorporatedby the polymerase are degraded. This allows the sequencing procedure toproceed without washing the template between successive nucleotideadditions. A further advantage is that since washing steps are avoided,it is not necessary to add new enzymes eg. polymerase with each newnucleotide addition, thus improving the economy of the procedure. Thus,the nucleotide-degrading enzyme or enzymes are simply included in thepolymerase reaction mix, and a sufficient time is allowed between eachsuccessive nucleotide addition for degradation of substantially most ofthe unincorporated nucleotides.

The amount of nucleotide-degrading enzyme to be used, and the length oftime between nucleotide additions may readily be determined for eachparticular system, depending on the reactants selected, reactionconditions etc.

As mentioned above, the nucleotide-degrading enzyme(s) may be includedduring the polymerase reaction step. This may be achieved simply byadding the enzyme(s) to the polymerase reaction mixture prior to,simultaneously with or after the polymerase reaction (ie. the chainextension or nucleotide incorporation) has taken place, e.g. prior to,simultaneously with, or after, the polymerase and/or nucleotides areadded to the sample/primer.

In one embodiment, the nucleotide-degrading enzyme(s) may simply beincluded in solution in a reaction mix for the polymerase reaction,which may be initiated by addition of the polymerase or nucleotide(s).Alternatively, the nucleotide-degrading enzyme(s) may be immobilized ona solid support, e.g. a particulate solid support (e.g. magnetic beads)or a filter, or dipstick etc. and it may be added to the polymerasereaction mixture at a convenient time. For example, such immobilizedenzyme(s) may be added after nucleotide incorporation (i.e., chainextension) has taken place, and then, when the incorporated nucleotidesare hydrolyzed, the immobilized enzyme may be removed from the reactionmixture (e.g. it may be withdrawn or captured, e.g., magnetically in thecase of magnetic beads), before the next nucleotide is added. Theprocedure may then be repeated to sequence more bases.

Such an arrangement has the advantage that more efficient nucleotidedegradation may be achieved as it permits more nucleotide degradingenzyme to be added for a shorter period. This arrangement may alsofacilitate optimization of the balance between the two competingreactions of DNA polymerization and nucleotide degradation.

In a further embodiment, the immobilization of the nucleotide-degradingenzyme may be combined with the use of the enzyme(s) in solution. Forexample, a lower amount may be included in the polymerase reactionmixture and, when necessary, nucleotide-degrading activity may beboosted by adding immobilized enzyme as described above. The termdideoxynucleotide as used herein includes all 2′-deoxynucleotides inwhich the 3′-hydroxyl group is absent or modified and thus, while ableto be added to the primer in the presence of the polymerase, is unableto enter into a subsequent polymerization reaction.

The method of the invention may readily be modified to enable thesequencing (ie. base incorporation) reactions to be continuouslymonitored in real time. This may simply be achieved by performing thechain extension and detection, or signal-generation, reactionssubstantially simultaneously by monitoring a detectable property of thetags on the NTPs during binding, incorporation, and/or pyrophosphaterelease.

The sample DNA (i.e., DNA template) may conveniently be single-stranded,and may either by immobilized on a solid support or in solution. The useof a nucleotide degrading enzyme according to the present inventionmeans that it is not necessary to immobilize the template DNA tofacilitate washing, since a washing step is no longer required. By usingthermostable enzymes, double-stranded DNA templates might also be used.The sample DNA may be provided by any desired source of DNA, includingfor example PCR or other amplified fragments, inserts in vectors such asM13 or plasmids.

In order to repeat the method cyclically and thereby sequence the sampleDNA and, also to aid separation of a single stranded sample DNA from itscomplementary strand, the sample DNA may optionally be immobilized orprovided with means for attachment to a solid support. Moreover, theamount of sample DNA available may be small and it may therefore bedesirable to amplify the sample DNA before carrying out the methodaccording to the invention.

The sample DNA may be amplified, and any method of amplification may beused, for example in vitro by PCR, rolling circle, or Self SustainedSequence Replication (3SR) or in vivo using a vector and, if desired, invitro and in vivo amplification may be used in combination. Whichevermethod of amplification is used the procedure may be modified that theamplified DNA becomes immobilized or is provided with means forattachment to a solid support. For example, a PCR primer may beimmobilized or be provided with means for attachment to a solid support.Also, a vector may comprise means for attachment to a solid supportadjacent the site of insertion of the sample DNA such that the amplifiedsample DNA and the means for attachment may be excised together.

Immobilization of the amplified DNA may take place as part of PCRamplification itself, as where one or more primers are attached to asupport, or alternatively one or more of the PCR primers may carry afunctional group permitting subsequent immobilization, eg. a biotin orthiol group. Immobilization by the 5′ end of a primer allows the strandof DNA emanating from that primer to be attached to a solid support andhave its 3′ end remote from the support and available for subsequenthybridization with the extension primer and chain extension bypolymerase.

The solid support may conveniently take the form of microtitre wells,which are advantageously in the conventional 8×12 format, or dipstickswhich may be made of polystyrene activated to bind the primer DNA (KAlmer, Doctoral Theses, Royal Institute of Technology, Stockholm,Sweden, 1988). However, any solid support may conveniently be used,including any of the vast number described in the art, e.g., forseparation/immobilization reactions or solid phase assays. Thus, thesupport may also comprise particles, fibers or capillaries made, forexample, of agarose, cellulose, alginate, Teflon or polystyrene.Magnetic particles eg the superparamagnetic beads produced by Dynal AS(Oslo, Norway) also may be used as a support.

The solid support may carry functional groups such as hydroxyl,carboxyl, aldehyde or amino groups, or other moieties such as avidin orstreptavidin, for the attachment of primers. These may in general beprovided by treating the support to provide a surface coating of apolymer carrying one of such functional groups, e.g. polyurethanetogether with a polyglycol to provide hydroxyl groups, or a cellulosederivative to provide hydroxyl groups, a polymer or copolymer of acrylicacid or methacrylic acid to provide carboxyl groups or an aminoalkylatedpolymer to provide amino groups. U.S. Pat. No. 4,654,267 describes theintroduction of many such surface coatings.

Another aspect of this invention is the use of tagged nucleotides todirectly sequence populations of DNA molecules in synchrony. Thesynchrony is achieved by applying a population of single tagged dNTP tothe reaction chamber, monitoring PP_(i) release (as per tag detection),and removing unincoporated dNTPs prior to applying the next tagged dNTPuntil the sequence of interest in determined.

The tagged NTPs and/or pyrophosphate detection method of the presentinvention thus opens up the possibility for an automated approach forlarge-scale, non-elecrophoretic sequencing procedures, which allow forcontinuous measurement of the progress of the polymerization reactionwith time. The method of the invention also has the advantage thatmultiple samples may be handled in parallel. The target DNA may be cDNAsynthesized from RNA in the sample and the method of the invention isthus applicable to diagnosis on the basis of characteristic RNA. Suchpreliminary synthesis can be carried out by a preliminary treatment witha reverse transcriptase, conveniently in the same system of buffers andbases of subsequent PCR steps if used. Since the PCR procedure requiresheating to effect strand separation, the reverse transcriptase will beinactivated in the first PCR cycle. When mRNA is the sample nucleicacid, it may be advantageous to submit the initial sample, e.g. a serumsample, to treatment with an immobilized polydT oligonucleotide in orderto retrieve all mRNA via the terminal polyA sequences thereof.Alternatively, a specific oligonucleotide sequence may be used toretrieve the RNA via a specific RNA sequence. The oligonucleotide canthen serve as a primer for cDNA synthesis, as described in WO 89/0982.Of course, the methods of the present invention can be used with anysource of purified cDNA and RNA.

The present invention also relates to using a modified nucleotide toincrease fidelity either alone (in a reaction with Taq DNA polymerase orany enzyme that joins nucleic acid monomers) or in combination with anaturally occurring, high-fidelity polymerase or one that is geneticallymodified for high-fidelity synthesis. Additionally, the inventors havedesigned a complementary system using tagged nucleotides, either aloneor in combination, with a naturally occurring low-fidelity polymerase orone that is genetically modified for low-fidelity synthesis. The purposeof this embodiment is to enable either random mutagenesis of aparticular nucleic acid or targeted mutagenesis of a particular basetype alone the length of the nucleic acid polymer. At times, it isdesirable to synthesize a nucleic acid polymer at reduced accuracy(essentially a random mutagenesis). In this system, a single or a subsetof natural nucleotides (that produced reduced fidelity synthesis), orratio thereof, can be used to more precisely target mutagenesis ofdesired nucleotide type along the length of the nucleic acid polymer.

Advantageously, the extension primer is sufficiently large to provideappropriate hybridization with the sequence immediately 5′ of the targetposition, yet still reasonably short in order to avoid unnecessarychemical synthesis. It will be clear to persons skilled in the art thatthe size of the extension primer and the stability of hybridization willbe dependent to some degree on the ratio of A-T to C-G base pairings,since more hydrogen bonding is available in a C-G pairing.

The present invention also relates to improving polymerase baseincorporation fidelity acting on a target nucleic acid sequence to whicha primer library (pre-existing primer set), where the library comprisesan optimized subset of base permutations and combinations of the fourbases for a base length between about 6 and about 35 bases. Theinventors have developed a robust “classic” sequencing strategy usingoctamer primers to initiate the DNA synthesizing reaction (Hardin et al.U.S. Pat. No. 6,083,695, incorporated herein by reference) The primerscomprising an octamer library are also appropriate to initiate singlemolecule DNA sequencing and related techniques with the modifiednucleotides. As an example, by contacting a target nucleic acid sequencewith such a library, complementary library primers will bind to thesequence forming a site for polymerase binding and polymerization. Ifmore than one primer binds, then the polymerase will randomly select andbind to a given primer complemented to a local on the sequence andpolymerization will commence. Although it is possible to have two primermolecules bound on a single template, only one primer will be the siteof polymerase activity. The increased fidelity will ensure superiorlibrary differentiation. Alternatively, the library members can containa 5′ extension to enable their immobilization to a surface, while stillretaining the ability to form at least an 8 base duplex with thetemplate, the unknown nucleic acid sequence (template) added to thesurface and then the polymerase and polymerizing components are added toinitiate polymerization with improved fidelity through the tagged dNTPsor by the addition of a phosphorolysis inhibitors of this invention.

Also, the skilled person will consider the degree of homology betweenthe extension primer to other parts of the amplified sequence and choosethe degree of stringency accordingly. Guidance for such routineexperimentation can be found in the literature, for example, MolecularCloning: a laboratory manual by Sambrook, J., Fritsch E. F. andManiatis, T. (1989). It may be advantageous to ensure that thesequencing primer hybridizes at least one base inside from the 3′ end ofthe template to eliminate blunt-ended DNA polymerase activity. Ifseparate aliquots are used (i.e., 4 aliquots, one for each base), theextension primer is preferably added before the sample is divided intofour aliquots although it may be added separately to each aliquot. Itshould be noted that the extension primer may be identical with the PCRprimer but preferably it is different, to introduce a further element ofspecificity into the system.

Alternatively, can have multiple (individual oligonucleotides such asoctamers) on surface where polymerization starts when a polymerizingagent such as a polymerase and the reaction components are added.

Additionally, primase may be used to synthesize an RNA primer that cansubsequently be used by a DNA polymerase to begin DNA synthesis. Thesite of initiation of the RNA chain is not critical and many reactionscan be processed in parallel to obtain the complete DNA sequence.

The polymerase reaction in the presence of the extension primer and adeoxynucleotide is carried out using a polymerase which will incorporatedideoxynucleotides, e.g. T7 polymerase, Klenow or Sequenase Ver. 2.0(USB U.S.A.). Any suitable chain extension sometimes are digested by anexonuclease activity. If such reverse polymerization occurs in themethod according to the invention the level of background noiseincreases. In order to avoid this problem, a nonproofreading polymerase,eg. exonuclease deficient (exo−) Klenow polymerase may be used.Otherwise it is desirable to add fluoride ions or nucleotidemonophosphates which suppress 3′ digestion by polymerase. The precisereaction conditions, concentrations of reactants etc. may readily bedetermined for each system according to choice. However, it may beadvantageous to use an excess of polymerase over primer/template toensure that all free 3′ ends are extended.

In the method of the invention there is a need for a DNA polymerase withhigh efficiency in each extension step due to the rapid increase ofbackground signal which may take place if templates which are not fullyextended accumulate. An induced-fit binding mechanism in thepolymerization step selects very efficiently for binding of the correctdNTP with a net contribution towards fidelity of 10-10⁶. Exonucleasedeficient polymerases, such as (exo-) Klenow or Sequenase 2.0, catalyzeincorporation of a nucleotide only when the complementary dNTP waspresent, confirming a high fidelity of these enzymes even in the absenceof proof-reading exonuclease activity.

In certain circumstances, e.g. with longer sample templates, it may beadvantageous to use a polymerase which has a lower Km for incorporationof the correct (matched) nucleotide, than for the incorrect (mismatched)nucleotide. This may improve the accuracy and efficiency of the method.

In many diagnostic applications, for example genetic testing forcarriers of inherited disease, the sample will contain heterozygousmaterial, that is half the DNA will have one nucleotide at the targetposition and the other half will have another nucleotide. Thus if fouraliquots are used in an embodiment according to the invention, two willshow a negative signal and two will show half the positive signal. Itwill be seen therefore that it is desirable to quantitatively determinethe amount of signal detected in each sample.

Also, it will be appreciated that if two or more of the same base areadjacent the 3′-end of the primer a larger signal will be produced. Inthe case of a homozygous sample it will be clear that there will bethree negative and one positive signal when the sample is in fouraliquots.

Further to enhance accuracy of the method, bidirectional sequencing ie.sequencing of both strands of a double-stranded template may beperformed. This may be advantageous e.g. in the sequencing ofheterozygous material. Conveniently, this may be achieved byimmobilizing the double-stranded sample template by one strand, e.g. onparticles or in a microtitre well, eluting the second strand andsubjecting both strands separately to a sequencing reaction by themethod of the invention.

Reaction efficiency may be improved by including Mg²⁺ ions in thereagent (NTP and/or polymerase) solutions. It will be appreciated thatwhen the target base immediately 3′- of the primer has an identical base3′- thereto, and the polymerization is effected with a deoxynucleotide(rather than a dideoxynucleotide) the extension reaction will add twobases at the same time and indeed any sequence of successive identicalbases in the sample will lead to simultaneous incorporation ofcorresponding bases into the primer. However, the amount ofpyrophosphate liberated will clearly be proportional to the number ofincorporated bases so that there is no difficulty in detecting suchrepetitions. Since the primer is extended by a single base by theprocedure described above (or a sequence of identical bases), theextended primer can serve in exactly the same way in a repeatedprocedure to determine the next base in the sequence, thus permittingthe whole sample to be sequenced.

As mentioned above, in the method of the invention, different taggeddeoxy- or dideoxynucleotides may be added to separate aliquots ofsample-primer mixture or successively to the same sample-primer mixture.This covers the situations where both individual and multiple target DNAsamples are used in a given reaction, which sample DNAs may be the sameor different. Thus, for example, as will be discussed in more detailbelow, in certain embodiments of the invention, there may be onereaction in one container, (in the sense of one sample DNA, ie. onetarget DNA sequence, being extended) whereas in other embodimentsdifferent primer-sample combinations may be present in the same reactionchamber, but kept separate by e.g. area-selective immobilization.

The present invention provides two principal methods of sequencingimmobilized DNA.

The invention provides a first method of sequencing sample DNA whereinthe sample DNA is subjected to amplification; the amplified DNA isoptionally immobilized and then subjected to strand separation, onestrand eg. the optionally non-immobilized or immobilized strand beingremoved (i.e., either strand may be sequenced), and an extension primeris provided, which primer hybridizes to the sample DNA immediatelyadjacent that portion of the DNA to be sequenced; each of four aliquotsof the single stranded DNA is then subjected to a polymerase reaction inthe presence of a tagged deoxynucleotide, each aliquot using a differenttagged deoxynucleotide whereby only the tagged deoxynucleotidecomplementary to the base in the target position becomes incorporated;the tagged pyrophosphate released by base incorporation beingidentified. After identification of the incorporated nucleotide anucleotide degrading enzyme is added, e.g., a phosphatase such as snakevenom phosphatase, calf intestinal phosphatase, shrimp alkalinephosphatase, or bacterial alkaline phosphatase. Upon separating thenucleotide degrading enzyme from the different aliquots, for example ifit is immobilized on magnetic beads, the four aliquots can be used in anew cycle of nucleotide additions, only if the other three are alsoextended by addition of the correct dNTP (after which, only one could beextended). This procedure can then be continuously repeated.

The invention also provides a second method of sequencing sample DNAwherein the sample DNA is subjected to amplification; the amplified DNAis optionally immobilized and then subjected to strand separation, onestrand, e.g., the optionally non immobilized or immobilized strand beingremoved, and an extension primer is provided, which primer hybridizes tothe sample DNA immediately adjacent to that portion of the DNA to besequenced; the single stranded DNA is then subjected to a polymerasereaction in the presence of a first tagged deoxynucleotide, and theextent of tagged pyrophosphate release is determined, non-incorporatednucleotides being degraded by the nucleotide-degrading enzyme, and thereaction being repeated by successive addition of a second, third andfourth tagged deoxynucleotide until a positive release of pyrophosphateindicates incorporation of a particular tagged deoxynucleotide into theprimer, whereupon the procedure is repeated to extend the primer onebase (or one base-type) at a time and to determine the base which isimmediately 3′- of the extended primer at each stage.

The present invention also provides a step by step polymerizationapparatus and method. The apparatus and method includes a tubular memberincluding a zone containing an immobilized polymerizing agent such as apolymerase. The zone is pre-treated by a nucleotide sequence. A plug ofsolution containing a polymerization reaction mixture containing taggeddNTPs, where each dNTP has a different tag having a different value fora specific detectable property such as absorption coefficient orfluoresceing frequency and where the tag are bonded to or associatedwith the β and/or γ phosphate of the triphosphate moiety. After aspecified period of time, a plug of inert buffer is moved into thetubular member to displace the reactive solution. As the reactivesolution is moved out of the reaction zone, the solution is exposed to adetection procedure that detects the specific detectable property todetermine the incorporated dNTP. The wash plug should generally beseveral multiples of the volume of solution necessary to cover thereaction zone. Another reactive medium plug then follows, followed by awash plug which allows detection of the next incorporation and so on andso on. Alternatively, the reaction media can include only a single dNTPand the detector looks for free tagged pyrophosphate. Each of thesestep-by-step polymerization methods is amenable of microarrayconfiguration. Additionally, for the single dNTP apparatus and method,combinatorial mathematics can be used to determine the best choice forthe next dNTP plug to use.

An alternative format for the analysis is to use an array format whereinsamples are distributed over a surface, for example a micro-fabricatedchip, and thereby an ordered set of samples may be immobilized in a 2dimensional (2D) format. Many samples can thereby be analyzed inparallel. Using the method of the invention, many immobilized templatesmay be analyzed in this way by allowing the solution containing theenzymes and one nucleotide to flow over the surface and then detectingthe signal produced for each sample. This procedure can then berepeated. Alternatively, several different oligonucleotidescomplementary to the template may be distributed over the surfacefollowed by hybridization of the template. Incorporation of taggeddeoxynucleotides or tagged dideoxynucleotides may be monitored for eacholigonucleotide by the signal produced using the variousoligonucleotides as primer. By combining the signals from differentareas of the surface, sequence-based analyses may be performed by fourcycles of polymerase reactions using the various taggeddideoxynucleotides.

Two-stage PCR (using nested primers), as described in applicationWO90/11369, may be used to enhance the signal to noise ratio and therebyincrease the sensitivity of the method according to the invention. Bysuch preliminary amplification, the concentration of target DNA isgreatly increased with respect to other DNA which may be present in thesample and a second-stage amplification with at least one primerspecific to a different sequence of the target DNA significantlyenhances the signal due to the target DNA relative to the ‘backgroundnoise’.

Any suitable polymerase may be used, although it is preferred to use athermophilic enzyme such as Taq polymerase to permit the repeatedtemperature cycling without having to add further polymerase, e.g.Klenow fragment, in each cycle of PCR. PCR has been discussed above as apreferred method of initially amplifying target DNA although the skilledperson will appreciate that other methods may be used instead of incombination with PCR. A recent development in amplification techniqueswhich does not require temperature cycling or use of a thermostablepolymerase is Self Sustained Sequence Replication (3SR) or rollingcircle amplification. 3SR is modelled on retroviral replication and maybe used for amplification (see for example Gingeras, T. R. et al PNAS(USA) 87:1874-1878 and Gingeras, T. R. et al PCR Methods andApplications Vol. 1, pp 25-33). Rolling circle amplification is known inthe art.

As indicated above, the method can be applied to identifying the releaseof tagged pyrophosphate when dideoxynucleotide residues are incorporatedinto the end of a DNA chain. The present invention also relates to amethod of identification of the base in a single target position in aDNA sequence (mini-sequencing) wherein sample DNA is subjected toamplification; the amplified DNA is immobilized and then subjected tostrand separation, the non-immobilized strand being removed and anextension primer, which hybridizes to the immobilized DNA immediatelyadjacent to the target position, is provided; each of four aliquots ofthe immobilized single stranded DNA is then subjected to a polymerasereaction in the presence of a dideoxynucleotide, each aliquot using adifferent dideoxynucleotide whereby only the dideoxynucleotidecomplementary to the base in the target position becomes incorporatedand, because the tagged nucleotides improve the fidelity ofincorporation, the signals from the other nucleotides are at background;the four aliquots are then subjected to extension in the presence of allfour deoxynucleotides, whereby in each aliquot the DNA which has notreacted with the dideoxynucleotide is extended to form double strandedDNA while the dideoxyblocked DNA remains as single stranded DNA;followed by identification of the double stranded and/or single strandedDNA to indicate which dideoxynucleotide was incorporated and hence whichbase was present in the target position. Clearly, the release of taggedpyrophosphate in the chain terminating dideoxynucleotide reaction willindicate which base was incorporated but the relatively large amount oftagged pyrophosphate released in the subsequent deoxynucleotide primerextension reactions (so-called chase reactions) gives a much largersignal and is thus more sensitive.

It will usually be desirable to run a control with no dideoxynucleotidesand a ‘zero control’ containing a mixture of all fourdideoxynucleotides. WO93/23562 defines the term ‘dideoxynucleotide’ asincluding 3′-protected 2′-deoxynucleotides which act in the same way bypreventing further chain extension. However, if the 3′ protecting groupis removable, for example by hydrolysis, then chain extension (by asingle base) may be followed by unblocking at the 3′ position, leavingthe extended chain ready for a further extension reaction. In this way,chain extension can proceed one position at a time without thecomplication which arises with a sequence of identical bases, asdiscussed above.

Thus, the methods A and B referred to above can be modified whereby thebase added at each stage is a 3′protected 2′-deoxynucleotide and afterthe base has been added (and the tag signal or detectable property suchas light emission is detected), the 3′-blocking group is removed topermit a further 3′-protected-2′ deoxynucleotide to be added. Suitableprotecting groups include acyl groups such as alkanol groups e.g. acetylor indeed any hydroxyl protecting groups known in the art, for exampleas described in Protective Groups in Organic Chemistry, JFW McOnie,Plenum Press, 1973.

The invention, in the above embodiment, provides a simple and rapidmethod for detection of single base changes. In one format itsuccessfully combines two techniques: solid-phase technology (DNA boundto magnetic beads) and detection of a detectable property associatedwith the tags on the dNTPs or released PP_(i). The method can be used toboth identify and quantitate selectively amplified DNA fragments. It canalso be used for detection of single base substitutions and forestimation of the heterozygosity index for an amplified polymorphic genefragment. This means that the method can be used to screen for rarepoint mutations responsible for both acquired and inherited diseases,identify DNA polymorphisms, and even differentiate betweendrug-resistant and drug-sensitive strains of viruses or bacteria withoutthe need for centrifugations, filtrations, extractions orelectrophoresis. The simplicity of the method renders it suitable formany medical (routine analysis in a wide range of inherited disorders)and commercial applications.

The positive experimental results presented below clearly show themethod of the invention is applicable to an on-line automaticnon-electrophoretic DNA sequencing approach, with step-wiseincorporation of single deoxynucleotides. After amplification to yieldsingle-stranded DNA and annealing of the primer, thetemplate/primer-fragment is used in a repeated cycle of dNTPincubations. Samples are continuously monitored for a detectableproperty of the tagged PPi such as fluorescence. As the synthesis of DNAis accompanied by release of tagged pyrophosphate (PPi) in an amountequal to the amount of nucleotide incorporated, signals derived from thedetectable property of the tag are observed only when complementarybases are incorporated. Due to the ability of the method to determinePPi quantitatively, it is possible to distinguish incorporation of asingle base from two or several simultaneous incorporations. Since theDNA template is preferably obtained by PCR, it is relatively straightforward to increase the amount of DNA needed for such an assay.

As mentioned above our results open the possibility for a novel approachfor large-scale non-electrophoretic DNA sequencing, which allows forcontinuous determination of the progress of the polymerization reactionwith time. For the success of such an approach there is a need for highefficiency of the DNA polymerase due to the rapid increase of backgroundsignal if templates accumulate which are not “in phase”.

The new approach has several advantages as compared to standardsequencing methods. Firstly, the method is suitable for handling ofmultiple samples in parallel. Secondly, relatively cost-effectiveinstruments are envisioned. In addition, the method avoids the use ofelectrophoresis and thereby the loading of samples and casting of gels.A further advantage of the method of the present invention is that itmay be used to resolve sequences which cause compressions in thegel-electrophoretic step in standard Sanger sequencing protocols.

The method of the invention may also find applicability in other methodsof sequencing. For example, a number of iterative sequencing methods,advantageously permitting sequencing of double-stranded targets, basedon ligation of probes or adaptors and subsequent cleavage have beendescribed (see e.g. U.S. Pat. No. 5,599,675 and Jones, BioTechniques 22:938-946, 1997).

Such methods generally involve ligating a double stranded probe (oradaptor) containing a Class IIS nuclease recognition site to a doublestranded target (sample) DNA and cleaving the probe/adaptor-targetcomplex at a site within the target DNA, one or more nucleotides fromthe ligation site, leaving a shortened target DNA. The ligation andcleavage cycle is then repeated. Sequence information is obtained byidentifying one or more nucleotides at the terminus of the target DNA.The identification of the terminal nucleotide(s) may be achieved bychain extension using the method of the present invention.

Further to permit sequencing of a double stranded DNA, the method of theinvention may be used in a sequencing protocol based on stranddisplacement, e.g. by the introduction of nicks, for example asdescribed by Fu et al., in Nucleic Acids Research 1997, 25(3): 677-679.In such a method the sample DNA may be modified by ligating adouble-stranded probe or adaptor sequence which serves to introduce anick e.g. by containing a non- or mono-phosphorylated or dideoxynucleotide. Use of a strand-displacing polymerase permits a sequencingreaction to take place by extending the 3′ end of probe/adaptor at thenick, nucleotide incorporation being detected according to the method ofthe present invention.

The method of the invention may also be used for real-time detection ofknown single-base changes. This concept relies on the measurement of thedifference in primer extension efficiency by a DNA polymerase of amatched over a mismatched 3′ terminal. The rate of the DNA polymerasecatalyzed primer extension is measured by detection of the detectableproperty associated with the tag such as fluorescence as describedpreviously. In the single-base detection assay, single-stranded DNAfragments are used as template. Two detection primers differing with onebase at the 3′-end are designed; one precisely complementary to thenon-mutated DNA-sequence and the other precisely complementary to themutated DNA sequence. The primers are hybridized with the 3′-terminiover the base of interest and the primer extension rates are, afterincubation with DNA polymerase and deoxynucleotides, measured bydetecting the characteristics of the detectable property such asfluorescence of the tag. If the detection primer exactly matches to thetemplate a high extension rate will be observed. In contrast, if the3′-end of the detection primer does not exactly match to the template(mismatch) the primer extension rate will be much lower or eliminated bythe use of tagged dNTPs which increase fidelity via the addition oftagged PPi. The difference in primer extension efficiency by the DNApolymerase of a matched over a mismatched 3′-terminal can then be usedfor single-base discrimination. Thus, the presence of the mutated DNAsequence can be distinguished over the non-mutated sequence. Byperforming the assay in the presence of a nucleotide degrading enzyme,it is easier to distinguish between a match and a mismatch of the typethat are relatively easy to extend, such as T:G and C:T.

The invention also comprises kits for use in methods of the inventionwhich will normally include at least the following components: (a) atest specific primer which hybridizes to sample DNA so that the targetposition is directly adjacent to the 3′ end of the primer; (b) apolymerase; (c) an optional detection enzyme means for identifyingpyrophosphate release; (d) a nucleotide-degrading enzyme; (e)deoxynucleotides, or optionally deoxynucleotide analogues having amolecular and/or atomic tag bonded to or associated with a β- and/orγ-phosphate of the dNTP, optionally including, in place of dATP, a dATPanalogue which is capable of acting as a substrate for a polymerase butincapable of acting as a substrate for a said PPi-detection enzyme; and(f) optionally dideoxynucleotides, or optionally dideoxynucleotideanalogues, optionally γ-tagged ddATP being replaced by a ddATP analoguewhich is capable of acting as a substrate for a polymerase but incapableof acting as a substrate for a said PPi-detection enzyme.

If the kit is for use with initial PCR amplification then it will alsonormally include at least the following components: (i) a pair ofprimers for PCR, at least one primer having a means permittingimmobilization of said primer; (ii) a polymerase which is preferablyheat stable, for example Taq DNA polymerase; (iii) buffers for the PCRreaction; and (iv) tagged deoxynucleotides and/or tagged PPi (forincreased fidelity).

Single-Molecule DNA Sequencing System

Engineering a polymerase to function as a direct molecular sensor of DNAbase identity during base incorporation will significantly increase thespeed and utility of an enzymatic DNA sequencing system possible. Atthis point, direct readout from a polymerase to determine base sequencehas been described in U.S. Provisional Patent Application No. 60/216,594filed: Jul. 7, 2000. This sequencing system combines severalcutting-edge technologies, including single-molecule detection,fluorescent molecule chemistry, computational biochemistry, and geneticengineering of biomolecules.

The inventors have tested whether γ-phosphate modified dATP could beincorporated by DNA polymerase. Importantly, both biological activityand, unexpectedly, increased fidelity are associated with polymerizationof this γ-phosphate modified nucleotide. Since γ-phosphate modifieddNTPs are not commercially available, they are designed and synthesizedin Dr. Gao's lab. In the following section, the reaction route used toproduce the ANS-dATP is provided. This route is also provided as anexample for the synthesis of additional γ-tagged dNTPs.

Synthesis of γ-Phosphate Modified dNTPs

Yarbrough and co-workers reported the use of fluorescent nucleotides (Aand U) by DNA-dependent RNA polymerase (Yarbrough et al., 1979).Following these examples, the inventors synthesized a DNA version ofaminonaphthalene-1-sulfonate (ANS) γ-phosphoamide ATP. Specifically, ANSγ-phosphoamide dATP was synthesized as shown below:

Using diode array UV detection HPLC, the fraction containing the desiredproduct was easily identified by the distinct absorption of the ANSgroup at 366 nm. Additionally, ³¹P NMR spectra were recorded for theγ-phosphate tagged dATP and non-modified dATP in an aqueous solution.For each compound, three characteristic resonances were observed,confirming the triphosphate moiety in the γ-tagged dATP. The combinedanalyses—1H NMR, HPLC and UV spectra—provide supporting evidence for theformation of the correct compound.

Although ANS was used in this example, the tag can be any tag thatalters the fidelity of the polymerizing agent, exemplary examples ofsuch tags include alkyl groups having between 1 and 30 carbon atoms,aryl groups having between about 6 and about 40 carbon atoms, or alkaryland aralkyl groups having between about 7 and about 40 carbon atoms, ormixture or combinations thereof. The substituents can have any number ofhetero atoms in the structure provided the structure represents a stablemolecular system, where the hetero atoms including P, S, Si, N, O, orany other hetero atom that does not render the nucleotide toxic to thepolymerase. Exemplary examples include 4-aminophenol, 6-aminonaphthol,4-nitrophenol, 6-nitronaphthol, 4-methylphenol, 6-chloronaphthol,4-methoxyphenol, 6-bromonaphthol, 4-chlorophenol, 6-iodonaphthol,4-bromophenol, 4, 4′-dihydroxybiphenyl, 4-iodophenol,8-hydroxyquinoline, 4-nitronaphthol, 3-hydroxypyridine, 4-aminonaphthol,umbelliferone, 4-methylnaphthol, resorufin, 4-methoxynaphthol,8-hydroxypyrene, 4-chloronaphthol, 9-hydroxyanthracene, 4-bromonaphthol,6-nitro-9-hydroxyanthracene, 4-iodonaphthol, 3-hydroxyflavone,6-methylnaphthol, fluorescein, 6-methoxynaphthol, 3-hydroxybenzoflavone,1-hydroxy-2-propyne, 1-hydroxy-4-pentyne, 1-hydroxy-3-butyne,1-hydroxy-5-hexyne, Methanol, Ethanol, Propanol, Isopropanol, Butanol,Tert-butanol, Hexanol, Cyclohexanol, Heptanol, Octanol, Decanol,Undecanol, Dodecanol, 1-acetoxymethanol (CH30OCCH2-O-NTP),2-acetoxyethanol, 3-acetoxypropanol, 4-acetoxybutanol,5-acetoxypentanol, 6-acetoxyhexanol, 2-nitroethanol, 3-nitropropanol,4-nitrobutanol, 5-nitropentanol, 5-nitrohexanol, 1-hydroxy-3-propene,1-hydroxy-2-cyclohexene, 1-hydroxy-4-butene, 1-hydroxy-3-propaldehyde,1-hydroxy-5-pentene, 1-hydroxy-4-butanaldehyde, 1-hydroxy-6-hexene,1-hydroxy-3-Butanone, Phenol, 4-methyl-3-hydroxypyridine,4-Carboxyphenol, 5-methoxy-3-hydroxypyridine, 4-Acetoxymethylphenol,5-nitro-3-hydroxypyridine, 4-nitrophenol,5-acetoxymethyl-3-hydroxypyridine, 4-methylphenol,6-methyl-8-hydroxyquinoline, 4-methoxyphenol6-methoxy-8-hydroxyquinoline, 4-ethylphenol,4-methyl-8-hydroxyquinoline, 4-butylphenol, 6-nitro-8-hydroxyquinoline,naphthol, 4-acetoxymethyl-8-hydroxyquinoline, 4 or 6 or 8 methylnaphtholpyrene, 4 or 6 or 8 methoxynaphthol, 6-methyl-8-hydroxypyrene, 4 or 6 or8 nitronaphthol, 6-ethyl-8-hydroxypyrene, 4 or 6 or 8 ethylnaphthol,6-nitro-8-hydroxypyrene, 4 or 6 or 8 butylnaphthol6-(carboxysuccinimidylester) fluorescein, 4 or 6 or 8acetoxymethylnaphthol, 6-carboxymethyl-2, 7-dichlorofluorescein,Methanol Cyclohexanol, 2-carboxy ethanol, 3-carboxypropanol,4-carboxybutanol, 2-hydroxyethanol, 3-hydroxypropanol, 4-hydroxybutanol,2-aminoethanol, 2-nitroethanol, 3-aminopropanol, 3-nitropropanol,4-aminobutanol, 4-nitrobutanol, or any other similar substituent.Exemplary modified nucleotide include ANS modified nucleotide andAdenosine-5′-(γ-4-nitrophenyl) triphosphate,Adenosine-5′-(γ-4-iodonaphthyl), Guanosine-5′-(γ-4-nitrophenyl)triphosphate, triphosphate Adenosine-5′-(γ-6-methylnaphthyl)triphosphate, Cytosine-5′-(γ-4-nitrophenyl) triphosphate,Thymidine-5′-(γ-4-nitrophenyl) triphosphate,Adenosine-5′-(γ-6-methoxynaphthyl) triphosphate,Uracil-5′-(γ-4-nitrophenyl) triphosphate,3′-azido-3′-deoxythymidine-5′-(γ-4-nitrophenyl)triphosphate,Adenosine-5′-(γ-6-aminonaphthyl) triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-4-nitrophenyl)triphosphate,Adenosine-5′-(γ-6-nitronaphthyl) triphosphate,2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-4-nitrophenyl)triphosphate,Adenosine-5′-(γ-6-chloronaphthyl) triphosphate,Adenosine-5′-(γ-4-aminophenyl) triphosphate,Adenosine-5′-(γ-6-bromonaphthyl) triphosphate,Adenosine-5′-(γ-4-methylphenyl) triphosphate,Adenosine-5′-(γ-6-iodonaphthyl) triphosphate,Adenosine-5′-(γ-4-methoxyphenyl) triphosphate,Adenosine-5′-(γ-4′-hydroxybiphenyl) triphosphate,Adenosine-5′-(γ-4-chlorophenyl) triphosphate,Adenosine-5′-(γ-8-quinolyl) triphosphate, Adenosine-5′-(γ-4-bromophenyl)triphosphate, Adenosine-5′-(γ-3-pyridyl) triphosphate,Adenosine-5′-(γ-umbelliferone), Adenosine-5′-(γ-4-iodophenyl)triphosphate, Adenosine-5′-(γ-4-nitronaphthyl) triphosphate,Adenosine-5′-(γ-resorufin) triphosphate, Adenosine-5′-(γ-pyrene)triphosphate, Adenosine-5′-(γ-4-aminonaphthyl) triphosphate,Adenosine-5′-(γ-anthracene) triphosphate,Adenosine-5′-(Γ-6-nitroanthracene) triphosphate,Adenosine-5′-(γ-4-methylnaphthyl) triphosphate,Adenosine-5′-(γ-flavonyl) triphosphate,Adenosine-5′-(γ-4-methoxynaphthyl) triphosphate,Adenosine-5′-(γ-fluorescein) triphosphate, Adenosine-5′-(γ-benzoflavone)triphosphate, Adenosine-5′-(γ-4-chloronaphthyl) triphosphate,Adenosine-5′-(γ-(4-nitrophenyl)-γ′-(4-aminophenyl) triphosphate,Adenosine-5′-(γ-4-bromonaphthyl) triphosphate,Adenosine-5′-(γ-(4-nitrophenyl)-γ′-(4-nitronaphthyl) triphosphate,Adenosine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-acetoxypropyl)triphosphate, Guanosine-5′-(γ-methyl)triphosphate, Cytosine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-acetoxymethyl)triphosphate (CH30OCCH,—O-NTP),Thymidine-5′-(γ-methyl) triphosphate, Uracil-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-acetoxyethyl) triphosphate,3′-azido-3′-deoxythymidine-5-(γ-methyl)triphosphate,Adenosine-5′-(γ-acetoxybutyl)triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ, acetoxypentyl) triphosphate,2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-acetoxyhexyl) triphosphate, Adenosine-5′-(γ-ethyl)triphosphate, Adenosine-5′-(γ-2-nitroethyl) triphosphate,Adenosine-5′-(γ-propyl) triphosphate, Adenosine-5′-(γ-4-butyl)triphosphate, Adenosine-5′-(γ-3-nitropropyl) triphosphate,Adenosine-5′-(γ-hexyl) triphosphate, Adenosine-5′-(γ-octyl)triphosphate, Adenosine-5′-(γ-4-nitrobutyl)triphosphate,Adenosine-5′-(γ-decyl) triphosphate, Adenosine-5′-(γ-dodecyl)triphosphate, Adenosine-5′-(γ-5-nitropentyl)triphosphate,Adenosine-5′-(γ-isopropyl) triphosphate, Adenosine-5′-(γ-tert-butyl)triphosphate, Adenosine-5′-(γ-methyl)-(γ′-ethyl) triphosphate,Adenosine-5′-(γ-cyclohexyl) triphosphate,Adenosine-5′-(γ-methyl)-(γ′-propyl) triphosphate,Adenosine-5′-(γ-2-propenyl) triphosphate, Adenosine-5′-(γ-3-butenyl)triphosphate, Guanosine-5′-(γ-2-propenyl) triphosphate,Adenosine-5′-(γ-4-pentenyl) triphosphate, Cytosine-5′-(γ-2-propenyl)triphosphate, Adenosine-5′-(γ-5-hexenyl) triphosphate,Thymidine-5′-(γ-2-propenyl) triphosphate, Adenosine-5′-(γ-cyclohexenyl)triphosphate, Uracil-5′-(7-2-propenyl) triphosphate,Adenosine-5′-(γ-3-propanaldehyde) triphosphate,3′-azido-3′-deoxythymidine-5′-(γ-2-propenyl) triphosphate,Adenosine-5′-(γ-4-butanaldehyde) triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-2-propenyl) triphosphate,Adenosine-5′-(γ-3-butanone) triphosphate,2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-2-propenyl) triphosphate,Adenosine-5′-(γ-2-propynyl) triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-2-propynyl) triphosphate,Guanosine-5′-(γ-2-propynyl) triphosphate, Cytosine-5′-(γ-2-propynyl)triphosphate, 2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-2-propynyl)triphosphate Thymidine 5′-(γ-2-propynyl) triphosphate,Uracil-5′-(γ-2-propynyl) triphosphate, Adenosine-5′-(γ-3-butynyl)triphosphate, 3′-azido-3′-deoxythymidine-5′-(γ-2-propynyl) triphosphate,Adenosine-5′-(γ-4-pentynyl) triphosphate, Adenosine-5′-(γ-5-pentynyl)triphosphate, Adenosine-5′-(γ-4-phenyl) triphosphate, Adenosine-5′-(γ-(4or 6 or 8 acetoxymethyl naphthyl) triphosphate,Guanosine-5′-(γ-4-phenyl) triphosphate, Cytosine-5′-(γ-4-phenyl)triphosphate, Adenosine-5′-(γ-(4-methylpyridyl)triphosphate,Thymidine-5′-(γ-4-phenyl) triphosphate, Uracil-5′-(γ-4-phenyl)triphosphate, Adenosine-5′-(γ-(5-methoxypyridyl)triphosphate,3′-azido-3′-deoxythymidine-5′-(γ-4-phenyl) triphosphate,Adenosine-5′-(γ-(5-nitropyridyl)triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-4-phenyl) triphosphate,Adenosine-5′-(γ-(5-acetoxymethylpyridyl) triphosphate,2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-4-phenyl) triphosphate,Adenosine-5′-(γ-(6-methyl-1-quinolyl) triphosphate,Adenosine-5′-(γ-4-carboxyphenyl) triphosphate,Adenosine-5′-(γ-(6-methoxy-1-quinolyl)triphosphate,Adenosine-5′-(γ-(4-acetoxymethyl)phenyl) triphosphate,Adenosine-5′-(γ-(4-methyl-1-quinolyl)triphosphate,Adenosine-5′-(γ-4-nitrophenyl) triphosphate,Adenosine-5′-(γ-4-methylphenyl)triphosphate,Adenosine-5′-(γ-(6-nitro-1-quinolyl) triphosphate,Adenosine-5′-(γ-4-methoxyphenyl) triphosphate,Adenosine-5′-(γ-(4-acetoxymethylpyrenyl) triphosphate,Adenosine-5′-(γ-4-ethylphenyl) triphosphate,Adenosine-5′-(γ-(6-methylpyrenyl) triphosphate,Adenosine-5′-(γ-4-butylphenyl) triphosphate, Adenosine 5′-(γ-naphthyl)triphosphate, Adenosine-5′-(γ-(6-ethylpyrenyl) triphosphate,Adenosine-5′-(γ-(4 or 6 or 8 methyl naphthyl)triphosphate,Adenosine-5′-(γ-(6-nitropyrenyl) triphosphate, Adenosine-5′-(γ-(4 or 6or 8 methoxynaphthyl) triphosphate,Adenosine-5′-(γ-6-(carboxysuccinimidyl fluorescein) triphosphate,Adenosine-5′-(γ-(4 or 6 or 8 nitro naphthyl) triphosphate.Adenosine-5′-(γ-6-carboxymethyl-2, 7-dichlorofluorescein) triphosphate,Adenosine-5′-(γ-(4 or 6 or 8 ethyl naphthyl) triphosphate,Adenosine-5′-(γ-4-phenyl)-(γ′-4 nitrophenyl) triphosphate,Adenosine-5′-(γ-(4 or 6 or 8 butyl naphthyl)triphosphate,Adenosine-5′-(γ-4-phenyl)-(γ′-4 aminophenyl)triphosphate,Adenosine-5′-(γ-methyl) triphosphate, Adenosine-5′-(γ-3-aminopropyl)triphosphate, Guanosine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-4-aminobutyl) triphosphate, Cytosine-5′-(γ-methyl)triphosphate Adenosine-5′-(γ-cyclohexyl) triphosphate,Thymidine-5′-(γ-methyl) triphosphate Adenosine-5′-(γ-2-carboxyethyl)triphosphate, Uracil-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-3-carboxypropyl)triphosphate,3′-azido-3′-deoxythymidine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-4-carboxybutyl) triphosphate,3′-azido-2′,3′-dideoxythymidine-5′-(γ-methyl) triphosphate,Adenosine-5′-(γ-2-hydroxyethyl) triphosphate,2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ-methyl)triphosphate,Adenosine-5′-(γ-3-hydroxypropyl) triphosphate, Adenosine-5′-(γ-ethyl)triphosphate, Adenosine-5′-(γ-propyl) triphosphate,Adenosine-5′-(γ-4-hydroxybutyl) triphosphate, Adenosine-5′-(γ-4-butyl)triphosphate, Adenosine-5′-(γ-2-nitroethyl) triphosphate,Adenosine-5′-(γ-hexyl) triphosphate, Adenosine-5′-(γ-3-nitropropyl)triphosphate, Adenosine-5′-(γ-isopropyl) triphosphate,Adenosine-5′-(γ-4-nitrobutyl) triphosphate, Adenosine-5′-(γ-tert-butyl)triphosphate, Adenosine-5′-(γ-methyl)-(γ′-ethyl)triphosphate,Adenosine-5′-(γ-cyclohexyl) triphosphate,Adenosine-5′-(γ-2-aminoethyl)triphosphate,Adenosine-5′-(γ-methyl)-(γ′-propyl) triphosphate, or any other similarsubstituted nucleotide.

Polymerase Activity Assays Using Tagged dNTP(s)

The ability of a commercially available polymerase to incorporate thenovel dNTPs synthesized was monitored using primer extension assays.

TABLE I Primer Strand Definitions Used in Examples Primer Strand: TOP 5′GGT ACT AAG CGG CCG CAT G 3′ SEQ. ID 2 Template Strands: BOT-T 3′CCA TGA TTC GCC GGC GTA CT 5′ SEQ. ID 3 BOT-C 3′CCA TGA TTC GCC GGC GTA CC 5′ SEQ. ID 4 BOT-G 3′CCA TGA TTC GCC GGC GTA CG 5′ SEQ. ID 5 BOT-A 3′CCA TGA TTC GCC GGC GTA CA 5′ SEQ. ID 6 BOT-Sau 3′CCA TGA TTC GCC GGC GTA CCT SEQ. ID 7 AG 5′ BOT-TC 3′CCA TGA TTC GCC GGC GTA CTC SEQ. ID 8 5′ BOT-3TC 3′CCA TGA TTC GCC GGC GTA CTT SEQ. ID 9 TC 5′

‘TOP’ represents the primer strand of the DNA duplex molecules used inthe primer extension assays. Variants of the template strand arerepresented by ‘BOT’. The relevant feature of the DNA template isindicated after the hyphen. For example, BOT-T, BOT-C, BOT-G, BOT-A areused to monitor polymerase incorporation efficiency and fidelity foreither nucleotides or nucleotide variants of dATP, dGTP, dCTP, and dTTP,respectively.

γ-Phosphate-Tagged dNTP Incorporation by Taq Polymerase

The following example illustrates that commercially available Taq DNApolymerase efficiently incorporates the ANS-γ-phosphate dNTPs, thesyntheses and characterization of which are described above.

This first example illustrates the incorporation of ANS-γ-phosphate dATPto produce extended DNA products from primer/template duplexes. Thereactions were carried out in extension buffer and the resultingradiolabeled products were size separated on a 20% denaturingpolyacrylamide gel. Data were collected using a phosphorimaging system.Referring now FIG. 1, Lane 1 contained 5′ radiolabeled ‘TOP’ probe inextension buffer. Lane 2 contained Taq DNA polymerase, 50 μM dGTPincubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’).Lane 3 contained Taq DNA polymerase, 50 μM dATP incubated with a DNAduplex (radiolabeled TOP with excess ‘BOT-Sau’). Lane 4 contained TaqDNA polymerase, 50 μM ANS-γ-dATP incubated with a DNA duplex(radiolabeled TOP with excess ‘BOT-Sau’). Lane 5 contained Taq DNApolymerase, 50 μM dGTP incubated with a DNA duplex (radiolabeled TOPwith excess ‘BOT-TC’). Lane 6 contained spill-over from lane 5. Lane 7contained Taq DNA polymerase, 50 μM dATP incubated with a DNA duplex(radiolabeled TOP with excess ‘BOT-TC’). Lane 8 contained Taq DNApolymerase, 50 μM ANS-γ-dATP incubated with a DNA duplex (radiolabeledTOP with excess ‘BOT-TC’). Lane 9 contained Taq DNA polymerase, 50 μMdGTP incubated with a DNA duplex (radiolabeled TOP with excess‘BOT-3TC’). Lane 10 contained Taq DNA polymerase, 50 μM dATP incubatedwith a DNA duplex (radiolabeled TOP with excess ‘BOT-3TC’). Lane 11contained Taq DNA polymerase, ANS-γ-dATP incubated with a DNA duplex(radiolabeled TOP with excess ‘BOT-3TC’). Lane 12 contained 5′radiolabeled ‘TOP’ probe in extension buffer. Lane 13 contained 5′radiolabeled ‘TOP’ probe and Taq DNA polymerase in extension buffer.Oligonucleotide sequences are shown in Table 1.

Quantitative comparison of lane 1 with lane 4 demonstrates that verylittle non-specific, single-base extension was detected when ANS-γ-dATPwas included in the reaction, and the first incorporated base should bedGTP (which was not added to the reaction). Quantitative analysis oflanes 1 and 8 demonstrates that approximately 71% of the TOP primer areextended by a template-directed single base when ANS-γ-dATP was includedin the reaction and the first incorporated base should be dATP. Thus,Taq DNA polymerase incorporates γ-tagged nucleotides. Equally importantto the polymerase's ability to incorporate a γ-tagged nucleotide is itsability to extend the DNA polymer after the modified dATP wasincorporated. Comparison of lane 1 with lane 11 demonstrated that a DNAstrand was extended after a γ-tagged nucleotide was incorporated. Thus,incorporation of a modified nucleotide was not detrimental to polymeraseactivity. Note, too, that extension of the primer strand byincorporation of an ANS-γ-nucleotide depended upon Watson-Crickbase-pairing rules. In fact, the fidelity of nucleotide incorporationwas increased at least 15-fold by the addition of this tag to theγ-phosphate.

Analyzing the data from FIG. 1, the percentages for correct versusincorrect extension can be determined. Table II tabulates these result.

TABLE II Percent of Correct Extension versus Percent Incorrect ExtensionPercent Total Percent Mis- Percent Lane Descriptor Expected ResultCorrect Extended Extended 1 Background No Extension 89.91 10.09 10.9 2dGTP 1 base Extension 52.99 19.97 72.97 3 dATP No Extension 61.99 38.0138.01 4 g-dATP No Extension 87.43 12.57 12.57 5 dGTP No Extension 24.9975.01 75.01 6 Spill 7 dATP 1 base Extension 15.24 69.01 84.25 8 g-dATP 1base Extension 71.14 6.51 77.64 9 dGTP No Extension 32.20 67.80 67.80 10dATP 3 base Extension 27.11 54.92 82.03 11 g-dATP 3 base Extension 73.433.87 77.31 12 Background No Extension 95.19 4.81 4.81 13 Background NoExtension 95.92 4.08 4.08

From the data, the relative percent fidelity improvement can bedetermined of dATP and ANS-γ-phosphate tagged dATP. When G is to beincorporated and dATP is the only nucleotide in the reaction medium,then the tagged nucleotide provides about a 3 fold decrease inmisextensions. When a single A is to be incorporated and dATP is onlynucleotide in the reaction medium, then the tagged nucleotide providesabout an 11 fold decrease in misextensions. When three A's are to beincorporated and dATP is only nucleotide in the reaction medium, thenthe tagged nucleotide provides about a 14 fold decrease inmisextensions.

This next example illustrates the synthesis of extended DNA polymersusing all four ANS tagged γ-phosphate dNTPs. Products generated in thesereactions were separated on a 20% denaturing polyacrylamide gel, the gelwas dried and imaged following overnight exposure to a Fuji BAS 1000imaging plate. Referring now to FIG. 2, an image of (A) the actual gelimage, (B) a lightened phosphorimage and (C) an enhanced phosphorimage.Lane descriptions for A, B, and C follow: Lane 1 is the controlcontaining purified 10-base primer extended to 11 and 12 bases bytemplate-mediated addition of α-³²P dCTP. Lane 2 includes the sameprimer that was incubated with double-stranded plasmid DNA at 96° C. for3 minutes (to denature template). The reaction was brought to 37° C. (toanneal the primer to the template), Taq DNA polymerase and all fournatural dNTPs (100 μM, each) were added and the reaction was incubatedat 37° C. for 60 minutes. Lane 3 includes the same labeled primer thatwas incubated with double-stranded DNA plasmid at 96° C. for 3 minutes;Taq DNA Polymerase and all four γ-modified dNTPs (100 M, each) wereadded and the reaction was incubated at 37° C. for 60 minutes. Lane 4includes the control, purified 10-base primer that was extended to 11and 12 bases by the addition of α-³²P dCTP and was cycled in parallelwith the reactions in lanes 5-8. Lane 5 includes the same ³²P-labeledprimer that was incubated with double-stranded plasmid DNA at 96° C. for3 minutes, the reaction was brought to 37° C. for 10 minutes, duringwhich time Taq DNA polymerase and all four natural dNTPs (100 μM, each)were added. The reaction was cycled 25 times at 96° C. for 10 seconds,37° C. for 1 minute, and 70° C. for 5 minutes. Lane 6 includes the same³²P-labeled primer that was incubated with double-stranded plasmid DNAat 96° C. for 3 minutes, the reaction was brought to 37° C. for 10minutes, during which time Taq DNA polymerase and all four γ-modifieddNTPs (100 μM, each) were added. The reaction was cycled 25 times at 96°C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes. Lane 7includes nonpurified, 10-base, γ³²P-labeled primer that was incubatedwith double-stranded DNA plasmid at 96° C. for 3 minutes, the reactionwas brought to 37° C. for 10 minutes, during which time Taq DNApolymerase and all four natural dNTPs (100 μM, each) were added. Thereaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1minute, and 70° C. for 5 minutes. Lane 8 includes nonpurified, 10-base,γ³²P-labeled primer that was incubated with double-stranded DNA plasmidat 96° C. for 3 minutes, the reaction was brought to 37° C. for 10minutes, during which time Taq DNA polymerase and all four γ-modifieddNTPs (100 μM, each) were added. The reaction was cycled 25 times at 96°C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.Evident in the reactions involving tagged dNTPs is a substantialdecrease in pyrophosphorolysis as compared to reactions involvingnatural nucleotides.

This next example illustrates the synthesis of long DNA polymers usingall four ANS tagged γ-phosphate dNTPs. Each primer extension reactionwas split into two fractions, and one fraction was electrophoresedthrough a 20% denaturing gel (as described above), while the other waselectrophoresed through a 6% denaturing gel to better estimate productlengths. The gel was dried and imaged (overnight) to a Fuji BAS 1000imaging plate. Referring now to FIG. 3, an image of (A) the actual gel,(B) a lightened phosphorimage of the actual gel, and (C) an enhancedphosphorimage of the actual gel. Lane descriptions for A, B, and Cfollow: Lane 1 includes 123 Marker with size standards indicated at theleft of each panel. Lane 2 contained the control, purified 10-baseprimer extended to 11 and 12 bases by template-mediated addition ofα-^(32p) dCTP. Lane 3 contained the same ³²P-labeled primer that wasincubated with double-stranded plasmid DNA at 96° C. for 3 minutes (todenature template), the reaction was brought to 37° C. (to anneal theprimer to the template), Taq DNA polymerase and all four natural dNTPs(100 μM, each) were added and the reaction was incubated at 37° C. for60 minutes. Lane 4 includes the same ³²P-labeled primer that wasincubated with double-stranded DNA plasmid at 96° C. for 3 minutes, thereaction was brought to 37° C., Taq DNA polymerase and all fourγ-modified dNTPs (100 μM, each) were added and the reaction wasincubated at 37° C. for 60 minutes. Lane 5 includes the control,purified 10-base primer that was extended to 11 and 12 bases by theaddition of α-³²P-dCTP was cycled in parallel with the reactions inlanes 5-8. Lane 6 includes the same ³²P-labeled primer that wasincubated with double-stranded plasmid DNA at 96° C. for 3 minutes, thereaction was brought to 37° C. for 10 minutes, during which time Taq DNApolymerase and all four natural dNTPs (100 μM, each) were added. Thereaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1minute, and 70° C. for 5 minutes. Lane 7 includes the same ³²P-labeledprimer that was incubated with double-stranded plasmid DNA at 96° C. for3 minutes, the reaction was brought to 37° C. for 10 minutes, duringwhich time Taq DNA polymerase and all four γ-modified dNTPs (100 μM,each) were added. The reaction was cycled 25 times at 96° C. for 10seconds, 37° C. for 1 minute, and 70° C. for 5 minutes. Lane 8 includesnonpurified, 10-base, γ³²P-labeled primer that was incubated withdouble-stranded DNA plasmid at 96° C. for 3 minutes, the reaction wasbrought to 37° C. for 10 minutes, during which time Taq DNA polymeraseand all four natural dNTPs (100 μM, each) were added. The reaction wascycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70°C. for 5 minutes. Lane 9 includes nonpurified, 10-base, γ-³²P-labeledprimer that was incubated with double-stranded DNA plasmid at 96° C. for3 minutes, the reaction was brought to 37° C. for 10 minutes, duringwhich time Taq DNA polymerase and all four γ-modified dNTPs (100 μM,each) were added. The reaction was cycled 25 times at 96° C. for 10seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.

The majority of extension products in this reaction are several hundredbases long for both natural and γ-modified dNTPs, and a significantpercentage of these products are too large to enter the gel. Thus,demonstrating that the γ-phosphate tagged dNTPs are used by Taqpolymerase to generate long DNA polymers that are non-tagged or nativeDNA polymer chains.

Different Polymerases React Differently to they Modified Nucleotides

The indicated enzymes (Taq DNA Polymerase, DNA polymerase I—KlenowFragment, Pfu DNA Polymerase, HIV-1 Reverse Transcriptase, T7 DNAPolymerase Sequenase Version 2) were incubated in the manufacturerssuggested reaction buffer, 50 M of the indicated nucleotide were addedand the reactions, containing aDNA duplex (5′ radiolabeled TOP and thespecified template) were incubated at 37° C. for 30-60 minutes. Thereaction products were analyzed by size separation through a 20%denaturing gel.

Taq DNA polymerase efficiently uses the modified nucleotides tosynthesize extended DNA polymers at increased accuracy as shown in FIGS.1-6.

The Klenow fragment from E. coli DNA polymerase I efficiently usesγ-modified nucleotides, but does not exhibit the extreme fidelityimprovements observed with other enzymes as shown in FIG. 4.

Pfu DNA polymerase does not efficiently use γ-modified nucleotides andis, thus, not a preferred enzyme for the single-molecule sequencingsystem as shown in FIG. 5.

HIV-1 reverse transcriptase efficiently uses the γ-modified nucleotides,and significant fidelity improvement results as shown in FIG. 6.

Polymerization activity is difficult to detect in the reaction productsgenerated by native T7 DNA polymerase (due to the presence of theenzyme's exonuclease activity). However, its genetically modifiedderivative, Sequenase, shows that the γ-modified nucleotides areefficiently incorporated, and that incorporation fidelity is improved,relative to non-modified nucleotides. The experimental results fornative T7 DNA polymerase and Sequenase are shown in FIG. 7.

Thus, for Taq polymerase or HIV-1 reverse transcriptase, improvedfidelity, due to the use of the γ-modified dNTPs of this invention,enables single-molecule DNA sequencing. However, not all polymerasesequally utilize the γ-modified nucleotides of this invention,specifically, Klenow, Sequenase, HIV-1 reverse transcriptase and Taqpolymerases incorporate the modified nucleotides of this invention,while the Pfu DNA polymerase does not appear to incorporate (orincorporates very inefficiently) the modified nucleotides of thisinvention.

Elevated Temperature Affects the Stability of ANS-γ-Phosphate-TaggeddNTPs

This experiment illustrates the effect of elevated temperature onANS-tagged dNTPs. Specifically, γ-tagged dNTPs were heated for 7 minutesat 96° C. Primer extension reactions containing heat-treated oruntreated natural or ANS-tagged dNTPs were compared to determine theeffect of high temperature. The reactions were carried out in extensionbuffer and the resulting radiolabeled products were size separated on a20% denaturing polyacrylamide gel. Data were collected using aphosphorimaging system.

Referring to FIG. 8. Lane 1 contained free γ-³²P-labeled primer (‘TOP’).Lanes 2-9 are extension reactions containing the γ-³²P-labeled TOP thatwas annealed to a single-stranded template (‘BOT T6T’) at 96° C. for 3minutes (to form primer-template duplex). Taq DNA polymerase and thespecified dNTPs (10 μM) were added and the reactions were carried out at37° C. for 30 minutes. Each lane contained as follows: untreated naturaldATP (Lanes 2-3), heat-treated natural dATP (Lanes 4-5), untreatedANS-γ-tagged dATP (Lanes 6-7), heat-treated ANS-γ-tagged dATP (Lanes8-9). Lanes 10-17 are extension reactions containing the γ-³²P-labeledTOP that was annealed to a single-stranded template (‘BOT A6A’) at 96°C. for 3 minutes (to form primer-template duplex). Taq DNA polymeraseand the specified dNTPs (10 μM) were added and the reactions werecarried out at 37° C. for 30 minutes. Each lane contained as follows:untreated natural TTP (Lanes 10-11), heat-treated natural TTP (Lanes12-13), untreated ANS-γ-tagged TTP (Lanes 14-15), heat-treatedANS-γ-tagged TTP (Lanes 16-17).

Referring to FIG. 9. Lane 1 contained free γ-³²P-labeled primer (‘TOP’).Lanes 2-9 are extension reactions containing the γ-³²P-labeled TOP thatwas annealed to a single-stranded template (‘BOT G6G’) at 96° C. for 3minutes (to form primer-template duplex). Taq DNA polymerase and thespecified dNTPs (10 μM) were added and the reactions were carried out at37° C. for 30 minutes. Each lane contained as follows: untreated naturaldCTP (Lanes 2-3), heat-treated natural dCTP (Lanes 4-5), untreatedANS-γ-tagged dCTP (Lanes 6-7), heat-treated ANS-γ-tagged dCTP (Lanes8-9). Lanes 10-17 are extension reactions containing the γ-³²P-labeledTOP that was annealed to a single-stranded template (‘BOT C6C’) at 96°C. for 3 minutes (to form primer-template duplex). Taq DNA polymeraseand the specified dNTPs (10 μM) were added and the reactions werecarried out at 37° C. for 30 minutes. Each lane contained as follows:untreated natural dGTP (Lanes 10-11), heat-treated natural dGTP (Lanes12-13), untreated ANS-γ-tagged dGTP (Lanes 14-15), heat-treatedANS-γ-tagged dGTP (Lanes 16-17).

Comparison between the lanes containing untreated and heat-treatednatural dNTPs does not show significant, if any, change in terms ofextension patterns and amount of completely extended products. Incontrast, after heat-treatment the ANS-γ-tagged dNTPs behave more liketheir natural counterparts, indicating that the ANS-tag is heat-labile,which results in a possible loss thereof.

Temperature and Time of Extension can be Used to Modulate the Rate ofdNTP Incorporation by Taq Polymerase

This following example illustrates the effect of temperature and time onthe ability of Taq DNA Polymerase to produce extended DNA products fromprimer/template duplexes. The reactions were carried out in extensionbuffer and the resulting radiolabeled products were size separated on a10% denaturing polyacrylamide gel. Data were collected using aphosphorimaging system. Referring now to FIG. 10.

The reactions contain γ-³²P-labeled primer (‘TOP’) duplexed to asingle-stranded template (‘BOT-24’) [primer/template ratio—1:9], theappropriate buffer, Taq DNA polymerase and the specified nucleotides.Lanes 1-4 contain all four natural dNTPs (100 μM, each). The reactionswere carried out for 1 minute at temperatures ranging from 10 to 40° C.Lanes 5-8 contain all four ANS-γ-modified dNTPs (100 μM, each). Thereactions were carried out for 1 minute at temperatures ranging from 10to 40° C. Lanes 9-12 contain all four natural dNTPs (100 μM, each). Thereactions were carried out for 10 minutes at temperatures ranging from10 to 40° C. Lanes 13-16 contain all four ANS-γ-modified dNTPs (100 μM,each). The reactions were carried out for 10 minutes at temperaturesranging from 10 to 40° C. Lane 18 is a control containing 5′radiolabeled primer (‘TOP’) in extension buffer.

Quantitative comparison of lanes 1 through 4 and 5 through 8demonstrates that temperature affects the amount of completely extendedproduct when the reaction is carried out for 1 minute, regardless of thetype of dNTPs used (natural vs γ-modified dNTPs). The same is true forlanes 9 through 12 and 13 through 16 when the reaction duration is 10minutes. It is evident that time also affects the rate of polymerizationwith either types of nucleotides. Thus temperature and/or time can beused to modify polymerization rate of Taq DNA polymerase.

ANS γ-Phosphate-Modification of dNTPs Affects the Terminal TransferaseActivity of Taq DNA Polymerase

This example demonstrates that the addition of an ANS-γ-tag to naturaldNTPs affects the terminal transferase activity of commerciallyavailable Taq DNA Polymerase. The extension reactions were carried outin extension buffer at 37° C. for 30 minutes and the resultingradiolabeled products were size separated on a 10% denaturingpolyacrylamide gel. Data were collected using a phosphorimaging system.Referring now to FIG. 11. Lane 1 is a control reaction and contained TaqDNA Polymerase and DNA duplex (radiolabeled ‘TOP’ probe andsingle-stranded ‘BOT-24’ template at equal molar concentrations). Nonucleotides are added. Lane 2 contained Taq polymerase, DNA duplex andall four natural dNTPs (100 μM, each). Lane 3 contained Taq polymerase,DNA duplex and all four γ-modified dNTPs (100 μM, each).

Quantitative comparison of Lanes 2 and 3 demonstrates that in thereactions involving γ-modified dNTPs there is a substantial decrease inthe terminal transferase activity of Taq DNA polymerase. The majorextension product in Lane 2 is a result of this activity, while themajor extension product in Lane 3 is due to a template-directed additionof nucleotides. If, however, the presence of a non-templated base isdesirable or necessary for cloning or other purposes, it can be added byelevating the reaction temperature as shown in FIGS. 8 and 9 andallowing the heat treated nucleotides to act as substrates for theenzymes terminal transferase activity.

Summary of Polymerase Incorporation Results

Different Polymerases React Differently to the ANS-γ-modifiedNucleotides: primer extension reactions were performed to determine theability of various polymerases to incorporate γ-tagged dNTPs during DNApolymerization. Control reactions contained natural dNTPs to monitor fortemplate-directed nucleotide incorporation as well as formisincorporation as shown in FIG. 12. The reactions were performed inthe appropriate buffer and contained the specified polymerase,primer/template duplex (radiolabeled ‘TOP’ primer annealed to ‘BOT-3TC’template), and only the indicated dNTP. The reactions were carried outat room temperature or at 37° C. for 30 minutes and were stopped by theaddition of 0.5 mM EDTA. The volume of the reaction was then reduced toapproximately 2-4 μL, loading dye was added and the polymerizationproducts were electrophoresed through a 20% denaturing polyacrylamidegel. Arrows indicate the position of the free labeled ‘TOP’. Asterisksindicate 3-base extension.

From the data, the inventors have found that the ANS tag is thermallylabile. The fact can be used to allow the construction of DNA sequenceswith high fidelity and low fidelity regions. Thus, a DNA polymerizationcan be started at low temperature using ANS γ phosphate taggednucleotides until a give sequence length is attained (from a statisticalbasis) and then the temperature can be raised to liberate the ANS tagresulting in the extension of the sequence with lowered fidelity. Thereverse can be done by starting with lower fidelity (untagged)nucleotides, running the polymerization for a set time, destroying anyremaining untagged dNTPs with a phosphatase, and then adding the ANStagged dNTPs and polymerizing for a second set period. Optionally, themedium can then be heated to allow a second lower fidelity region to beprepared. Thus, the present invention can be used to prepare DNA, RNA ormixed sequences having high fidelity and low fidelity regions. Such DNA,RNA or mixed sequences can be used to investigate evolutionary trends,analyzing the mutagenecity of different regions of DNA sequences,producing nucleic acid polymers that contain both highly accurate andreduced accuracy regions (in any combination or order) for mutagenesisstudies (essentially targeted, random mutagenesis), or determine sitesprone to mutations that result in disease states, carcinogenic states orchange in cell phenotypes. The present invention also relates to methodfor preparing DNA, RNA or mixed sequences with regions of differentfidelity indices.

The present invention also relates to the following pyrophosphorolysisinhibitors selected from the group consisting of compounds of thefollowing general formulas or mixtures or combinations thereof:

Z—OPO₂O—Z′  (a)

Z—PO₂O—Z′  (b)

Z—OPO₂—Z′  (c)

Z—PO₂—Z′  (d)

Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)

Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)

Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)

Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h)

where Z or Z′ is a hydrogen atom or a thermally stable substituentcomprising primarily one or more atoms selected from the group carbon,nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atomsto satisfy valence requirements, E and E′ are an oxygen atom or athermally stable substituent comprising primarily one or more atomsselected from the group carbon, nitrogen, oxygen, sulfur and phosphoruswith sufficient hydrogen atoms to satisfy valence requirements and n isan integer having a value between 0 and about 5. The term primarilymeans that other atoms can be present, but in very small amounts.

The present invention relates to any nucleotide or nucleotide analogbearing a tag anywhere on the nucleotide (phosphate groups, base orsugar) that improves the fidelity of nucleotide incorporation.

Different DNA Polymerases React Differently to the γ-Phosphate ModifiedNucleotides

Primer extension experiments were performed with several different DNApolymerases to determine whether any of them could incorporate theANS-γ-phosphate modified nucleotides (FIG. 12). These experiments wereperformed by incubating the indicated polymerase in appropriateextension buffer, 100 μM ANS-γ-modified dATP, and a 5′-³²P end-labeledprimer annealed to a template that directed sequential incorporation ofdATP, dATP, dATP dGTP. Positive and negative control reactionscontaining natural dATP or dGTP, respectively, were run in parallel tomonitor template-directed nucleotide incorporation or mis-incorporation.Reactions containing dATP should produce 3-base extension products,whereas those containing dGTP should not produce extended products (dueto the absence of dATP). No products longer than 3-bases should result,since no reaction contained more than one nucleotide type. The reactionswere allowed to proceed for 30 minutes, at which time they were stoppedby EDTA addition, lyophilized and resuspended in 3.5 μl of sequencingloading buffer. Reaction products were heat denatured, loaded onto a 20%denaturing polyacrylamide gel, size-separated (2400 V; 1 hour), anddetected via phosphorimaging.

The inventors observed that Taq DNA polymerase I, HIV-1 ReverseTranscriptase (RT), Klenow fragment of E. coli DNA polymerase I, and amodified version of T7 DNA polymerase (Sequenase, Version 2) eachincorporate the modified nucleotides. Interestingly, the high-fidelityenzyme Pfu DNA polymerase appears least able to incorporate thesenucleotides. The inventors discovered that each polymerase respondeddifferently to the modified nucleotides. The fact that several differentDNA polymerases incorporate the ANS-γ-phosphate modified nucleotidesprovides critical feasibility data for the VisiGen Sequencing System.Note that the expected 3-base extension products accumulate in reactionscontaining ANS-γ-dATP, whereas the lanes containing natural dATP or dGTPproduce increased amounts of mis-extended products. Thus, the presenceof the ANS-modification on the γ-phosphate appears to increase theaccuracy of the reaction, and this may improve the accuracy of theVisiGen Sequencing System.

DNA Polymerase Efficiently Incorporates γ-Phosphate Modified Nucleotides

To begin to understand the unexpected observation that γ-modified dNTPsimprove the accuracy at which polymerases synthesize DNA strands, theinventors investigated the incorporation efficiency of ANS-γ-modifieddNTPs relative to their natural counterparts. These experiments wereperformed by incubating polymerase in extension buffer, 100 μM of theindicated natural or ANS-γ-modified dNTP, and a 5′-³²P end-labeledprimer (TOP)/single-stranded template (BOT-‘X’) duplex for 0.5, 1, 2, 3,5, 10 or 30 minutes. The sequences of the oligonucleotide templates areshown (Table 1). Intensities of non-extended primer band (I_(o)) versusextended primer band (I₁) were quantified with a Fuji MacBas1000 Imagesoftware version 3.0. To calculate the relative percent extension,background was first subtracted from each band value and the followingcalculation was applied to each reaction: Relative PercentExtension=((I₁)/(I₀+I₁))×100. The percent extensions observed in thetime course experiments are plotted and demonstrate that DNA polymeraseincorporates each ANS-γ-phosphate modified nucleotide at a similarefficiency as the corresponding natural nucleotide, thus providingadditional feasibility data for the VisiGen Sequencing System (FIG. 13).

γ-Phosphate Modified Nucleotides Improve Reaction Fidelity

Experiments demonstrating that commercially available Taq DNA polymeraseefficiently incorporates the modified nucleotides provides feasibilitydata for the VisiGen Sequencing System and, unexpectedly, datademonstrating that this modification increases the fidelity ofnucleotide incorporation (patent pending). In these experiments, Taq DNApolymerase (2.5 units/reaction; Promega Corporation) was incubated inpolymerase reaction buffer with 10,000 cpm of 5′-³²P end-labeled ‘TOP’primer, 10 ng of the indicated single-stranded template, and thespecified dNTP. The sequences of the oligonucleotides are shown (Table1). Extension reactions were incubated for 30 minutes, and terminated bythe addition of 1 μl of 0.5 M EDTA. The reactions were lyophilized andresuspended in 3.5 μl of sequencing loading buffer. Reaction productswere heat denatured, loaded onto a 20% denaturing polyacrylamide gel,size-separated, and quantified using a phosphorimaging system (FujiMedical Systems, Inc.).

A representative primer extension analysis that demonstrates thefidelity improvement is shown (FIG. 3). This is an important experimentbecause it illustrates the following:

1) Taq DNA Polymerase does not Randomly Incorporate γ-TaggedNucleotides.

Quantitative comparison of lane 1 with lane 4 demonstrates that verylittle non-specific, single-base extension is detected when ANS-γ-dATPis included in the reaction, but the first incorporated base should bedGTP (which was not added to the reaction). The ‘BOT-Sau’ template wasdesigned to monitor sequential incorporation of dGTP, dATP, dTTP anddCTP.

2) Taq DNA Polymerase Accurately Incorporates γ-Tagged Nucleotides.

Quantitative analysis of lanes 1 and 8 demonstrates that approximately70% of the TOP primer strands are extended by a template-directed singlebase when ANS-γ-dATP is included in the reaction and the firstincorporated base should be dATP. This percentage is very similar to thepercent extension observed with natural dATP (75%; lane 7). However, 60%of the extension products resulting from natural dATP incorporation weremisextended opposite a template C, and 34% of these products werefurther extended by the enzyme's terminal transferase activity.

3) DNA Strand Extension Continues Following γ-Tagged NucleotideIncorporation.

It was important to demonstrate that the polymerase could continueextension following incorporation of a γ-modified dNTP. This was firstaccomplished by preparing reactions containing the same end-labeled‘TOP’ primer hybridized with the ‘BOT-3TC’ template, and ANS-modifieddATP. Multiple occurrences of a single-base type in the extensiontemplate were used to simplify analysis of the extension products. Asingle nucleotide is added to the reaction and, thus, only thatnucleotide can be incorporated into the growing DNA strand. Comparisonof lane 1 with lane 11 demonstrates that multiple modified nucleotidesare incorporated and are, therefore, not detrimental to chain extension.Natural dATP (lane 10) is efficiently incorporated opposite Ts in thetemplate, but is also frequently misincorporated opposite a template C.Further, these blunt-ended molecules stimulate the enzyme's terminaltransferase activity and account for the formation of the 5 baseextension products.

4) Extension of the Primer Strand by Incorporation of anANS-γ-Nucleotide is Dependent Upon Watson-Crick Base Pairing Rules.

In fact, the fidelity of nucleotide incorporation is increased byγ-phosphate modification (patent pending).

Comparison of Relative Fidelity Improvement: Single Nucleotide ExtensionAssays

The inventors discovered that the fidelities of several commerciallyrelevant DNA polymerases are improved by providing the enzyme withnucleotides containing a molecular moiety at the γ-phosphate. This wasfurther investigated by assaying the percent extension of natural andγ-phosphate modified dNTP in complementary (matched) andnoncomplementary (mismatched) nucleotide combinations (FIG. 14). Inthese experiments, FIG. 14A was 5′ end-labeled, gel purified andquantified with regard to both radioactivity and absorbance at 260 nm.Primer/template hybrids were formed by heat denaturing and slow coolingprimer and template strands (1:1.2 ratio). Extension reactions wereprepared by combining the duplex with reaction buffer, dNTP or ANS-dNTPat 100 μM, and a DNA polymerase. To increase detection of mismatchincorporation, reactions were incubated for 30 minutes and terminated byadding STOP solution. Terminated reactions were heated, placed on ice,loaded onto a 20% denaturing polyacrylamide gel and electrophoresed for˜2 hours at 30 W. Gels were dried and imaged with a phosphorimagingsystem (Fuji Medical Systems, Inc.). Each reaction was repeated at leastthree times, and the average extension and average deviation werecalculated.

Interestingly, the magnitude of the fidelity improvement is influencedby the identity of the templating base versus the incoming nucleotide.As an example, C:T provides a different magnitude of improvement (15.98fold) when compared to T:C (2.93 fold), where the first base is thetemplate base and the second base is the incoming nucleotide. Comparingthe percent extension of a natural dNTP with that of an ANS-modifieddNTP, it is striking that the modified nucleotide is consistentlyincorporated at an improved accuracy. Additionally, the time coursestudies show that the incorporation of the complementary nucleotide(natural or ANS-modified) exhibit similar incorporation profiles,indicating that the fidelity improvements are not due to generallyslowed reaction kinetics resulting from nucleotide modification.

Kinetic analysis of the ANS-γ-phosphate fidelity affect is warranted inPhase II of the project since similar nucleotides will be used in theVisiGen Single-Molecule Sequencing System. It is likely that thesenucleotides will exhibit altered incorporation fidelities, similar tothe ANS-modified nucleotides. If increased fidelity is associated withincorporation of the fluorescently-modified nucleotides designed for theVisiGen Sequencing System, the accuracy of the single molecule sequencewill increase and the number of reactions that need to be performed inparallel to obtain highly accurate information will decrease.

The fidelity improving nucleotides—“Designer Nucleotides”—are beingpursued as a VisiGen intermediate product. We anticipate a shorter routeto this product. VisiGen's designer nucleotides will improve theaccuracy at which a DNA strand is synthesized and should be quite usefulin any enzymatic extension assay (patent pending). Perhaps derivativesof these nucleotides will enable highly accurate in vitro DNA synthesisthat rivals, or possibly exceeds, the accuracy at which a DNA strand isreplicated in vivo. Thus, Phase II of our project, “Real-time DNASequencing: Nucleotide Synthesis and Use”, will support kinetic analysisand M13 forward mutation assays of ANS-modified nucleotides. Data fromthese studies will define the ‘fidelity factor’ for each modifiednucleotide, and enable us to better understand the importance of themodification relative to the natural nucleotide. It is unlikely that thefirst molecular moiety chosen, ANS, is the one that produces the optimaldesigner nucleotide. Future studies (not supported by this award), willdefine the parameters that affect replication fidelity by examining thecontribution of specific tag modifications on matched versus mismatchednucleotide selection. However, the kinetic and forward mutation studiessupported by a Phase II award will provide feasibility data that willenable us to begin discussions with larger companies and/or privateinvestors interested in either a short- or long-term technology—designernucleotides that improve reaction fidelity and single-molecule DNAsequencing, respectively.

Chemically Engineer Nucleotides (NIH Phase II)

Synthesis of γ- and/or β-Modified and 3′-Modified dNTP

Potential candidate compounds for use in our FRET or quenching detectionof polymerase reactions are summarized in the following syntheticscheme:

In above scheme, N=A, C, G, T; X=O, N, S, CH₂, etc.; L=linker, such as—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—; R=fluorophore or quencher moieties, suchas ANS, FAM, FITC, rhodamine, cyanine, pyrene, perylene; P_(hv) is aphotolabile group.

These modified dNTPs can be used to identify a set of nucleotides thatwork with modified polymerase as an energy transfer pair to achieve highefficiency and accurate sequence reading. The positions that are ofinterest are the 5′-γ and 5′-β, since the modifications at thesepositions may not or least affect enzyme activity. The inventors plan toincorporate rhodamine derivatives, such as TMR or TAMRA, for FRETdetection and DABCYL for quenching detection (the enzyme in using eithertype of dNTP's will contain fluorescein moiety). An additional linker(L) between the dye moiety and phosphorous provides flexibility toobtain better fit of the nucleotide and the enzymatic active site andmay provide stabilization to the resultant modified compound. Thealteration at the phosphorous linkage is preferably an O (X=O). For all5′-modified dNTP's, if the 3′-OH is not blocked, the sequence willproceed continuously. The protection of the 3′-O position with aphotolabile group causes reaction to pause after the addition of a dNTP.The sequence extension can continue after photo-deprotection of the3′-P_(hv). The 3′-protection is to be used along with 5′-modificationsto overcome potential problems of background reading from fluorescentnucleotides.

Specifically, examples of the compounds the inventors plan to synthesizeare the following:

Set 1—these are 5′-γ or β-modifications, different dyes are needed sothat different nucleotides can be differentiated by differentwavelengths. These are the d-rhodamine family of molecules and have beenwidely used in traditional DNA sequencing. The linker and connector,HNCH₂CH₂O will be varied to achieve the best result.

AD-NHCH₂CH₂OP_(γ)P_(β)P_(α)-dN (N=A,C,G,T)

P_(γ)(AD-NHCH₂CH₂O)P_(β)P_(α)-dN (N=A,C,G,T)

where AD: acceptor dye molecule, AD is selected from Lee et al., 1997;AD=5dR110, 5dR6G, 5dTMR, 5dROX

Set 2—These are 5′-quencher (Q), 3′-P_(hv) modifications. Our laboratoryhas used this photolabile group in the synthesis of severalphotogenerated reagents, and have studied this reaction in great detail.

Q-NHCH₂CH₂OP_(γ)P_(β)P_(α)-dN-3′-OCO₂CH₂CH(CH₃)[(2-NO₂)Ph], (N=A,C,G,T)

P_(γ)(AD-NHCH₂CH₂O)P_(β)P_(α) dN-3′-OCO₂CH₂CH(CH₃)[(2-NO₂)Ph (N=A,C,G,T)

The inventors will closely monitor the results of our experiments andimplement changes in our synthesis plan as necessary.

The synthesis of γ-modified triphosphates will begin with typicalreactions for triphosphate esterification, using a coupling reagent suchas that shown in the Phase I Final Project Report section for thesynthesis of γ-ANS-dATP. Although the reaction is simple, the isolationof the product requires great care since the compound may not be quitestable. For separation of the ANS dNTP products, low temperature wasused and light must be kept away in the process. Rhodamine molecules arenot stable under basic conditions and caution will be taken. However,since the inventors have had long time experiences in synthesis andthere is a large of amount of information on the type of chemistry whichcan be used, the inventors do not anticipate significant problems. Thesynthesis of β-modified dNTP will require first protecting the activeγ-phosphate. The inventors do not intend to spend major effort in makingthese compounds, unless the β-modified molecule is a strong candidatewith desirable sequencing properties. To connect the linker with dye onone side and nucleotide on the other side, the inventors prefer to haveamide and phosphate bonds, respectively. The bond formation of thelinker is through well-known coupling reactions (through isothiocyannateester, or NHS ester, etc.).

All references cited herein are incorporated by reference. While thisinvention has been described fully and completely, it should beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described. Although theinvention has been disclosed with reference to its preferredembodiments, from reading this description those of skill in the art mayappreciate changes and modification that may be made which do not departfrom the scope and spirit of the invention as described above andclaimed hereafter.

1. A method for sequencing nucleic acids, comprising: a) flowing labeleddeoxynucleotide triphosphates onto template nucleic acids which arehybridized to a primer nucleic acid, where the template nucleic acids orprimers are attached to a support, and wherein the labeled nucleotidetriphosphates include a molecular tag bonded to the beta- orgamma-phosphate; b) incorporating one of the labeled nucleotidetriphosphates into at least one of the primers with a polymerase; c)releasing a tagged pyrophosphate which includes the molecular tag bondedto the beta- or gamma-phosphate; and d) detecting the released taggedpyrophosphates.
 2. The method of claim 1, further comprising: flowing instep (a) at least one compound selected from the group consisting ofcompounds of the following general formulas:Z—OPO₂—(OP(EE′)O)_(n)—PO₂O—Z′  (e)Z—OPO₂—(OP(EE′)O)_(n)PO₂—Z′  (f)Z—PO₂—(OP(EE′)O)_(n)PO₂O—Z′  (g)Z—PO₂—(OP(EE′)O)_(n)PO₂—Z′  (h) where Z and Z′ are each independently ahydrogen atom or a thermally stable substituent, E and E′ are eachindependently an oxygen atom or a thermally stable substituent and n isan integer having a value of 2, 3, 4 or
 5. 3. The method of claim 1,wherein the support comprises a micro-fabricated chip.
 4. The method ofclaim 1, wherein the template nucleic acids or the primers that arehybridized to the template nucleic acids are attached to the support inan ordered array.
 5. The method of claim 1, wherein the flowing I step(a) includes a single type of labeled deoxynucleotide triphosphatesselected from the group consisting of deoxyadenosine triphosphate,deoxycytosine triphosphate, deoxyguanosine triphosphate, deoxythymidinetriphosphate and deoxyuridine triphosphate.
 6. The method of claim 1,wherein the released tagged pyrophosphates are detected in parallel. 7.The method of claim 1, wherein the molecular tag comprisesaminonaphthalene-1-sulfonate (ANS).
 8. The method of claim 1, whereinthe molecular tag comprises an alkyl group, aryl group, alkaryl group,aralkyl group.
 9. The method of claim 1, wherein the released taggedpyrophosphate comprise a nitrogen or sulfur atom that substitutes forany of the oxygen atoms